Sensors for biomolecular detection and cell classification

ABSTRACT

A sensor device is provided for detecting an analyte in a sample in which an analyte is bound to a detection reagent to form a bound complex. The device comprises (a) a sample ( 5 ) comprising an ionic analyte and a detection reagent in a conductive fluid, wherein the detection reagent has a net charge different from the analyte; (b) a first permeable polymeric hydrogel plate ( 3 ) and a first spacer plate ( 8 ), which plates provide a compartment for the sample; (c) an anode ( 1 ) juxtaposed to the outside of the first hydrogel plate and not in contact with the sample; (d) a cathode ( 9 ) juxtaposed to the outside of the first spacer plate and not in contact with the sample; (e) a voltage generator ( 10 ) to apply an electric potential to the anode and cathode; and (f) a detector ( 11 ). The bound complex formed from the analyte and detection reagent is detected by the detector because the bound complex has a charge that causes it to migrate in a direction opposite from that of the unbound analyte when the electric potential is applied.

This application claims priority from PCT/US2003/031486, filed 3 Oct.2003, which is a continuation-in-part application of PCT/US2003/13538,filed 30 Apr. 2003, which application was filed as U.S. patent Ser. No.10/962,003 on 8 Oct. 2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to methods for detecting analytes suchas proteins, peptides, nucleic acids, ligands, antigens, lipids,enzymes, and other molecules in simple and complex systems.

2. Description of the Background

The disclosures referred to herein to illustrate the background of theinvention and to provide additional detail with respect to its practiceare incorporated herein by reference and, for convenience, arenumerically referenced in the following text and respectively grouped inthe appended bibliography.

A device that can be used to monitor gene expression rapidly in singlecells would have several important applications. For example, surgeonsoften rely on histological methods to distinguish tumor and normaltissues during surgery to remove cancers. These methods serve well whenthe morphology of the abnormal and normal cells is readilydistinguished. Unfortunately, the borders of many tumors are not alwayswell defined and do not provide clear landmarks that can be used toguide surgery. Further, it may be difficult to gauge the characteristicsof the tumor even after sections have been stained with histologicaldyes. This can lead to unnecessary surgery during efforts to remove allthe cancerous tissue. Indeed, some surgery for breast cancer involvesremoving lymph nodes to stage the cancer even though there often is noevidence that this additional surgery will be of significant benefit.Application of a technique that has the ability to monitor geneexpression in these frozen sections would have considerable applicationduring surgery to guide the procedure. It would also be useful to guidethe type of therapy that is to be used following surgery.

Recent advances in genetics have provided the basis by which physiciansand scientists have gained new insights into cell function.Bioinformatic analysis suggests that humans have 30-40 thousand genes[1;2] that are transcribed, spliced, and edited to yield 100 thousandmRNAs detected as expressed sequence tags [3]. This information haspermitted the design of microarrays capable of monitoring thousands ofgene products at one time [4;5]. Microarray technology is being appliedwidely to characterize changes in gene expression patterns that areassociated with various tumors and with the prognosis of tumor therapy[5-7]. Indeed, there is considerable hope that the results of thesestudies will enable a more accurate classification of tumors and therebyguide the choice of therapy following surgery. One benefit of this maybe a reduction in unnecessary chemotherapy or radiotherapy [5],procedures that often make patients ill and that may even be a source ofmalignancies later in life [8].

Further technical advances in measurements of gene expression productsare required to take full advantage of the new information that is beingmade available from microarray measurements. Tumors are often quitecomplex and contain endothelial cells, fibroblasts, lymphocytes, andother cell types in addition to transformed cells. Microarray analysesof whole tumor tissues detect expression products of these cell typessimultaneously [4;5], a phenomenon that confounds the association ofparticular gene expression patterns with specific tumor cells. Theseanalyses can be further compromised by the presence of different typesof tumor cells within the sample. Nonetheless, despite this complexity,gene expression patterns detected in some tumors are correlated highlywith five-year survival rates [5] and this information can be used tofacilitate tumor classification, the major parameter used to decide howpatients are treated.

The massive amount of data obtained during microarray analysis isextremely valuable but it is confounded by the presence of gene productsthat have been obtained from multiple cell types. It can also betime-consuming to obtain and, because it contains so much information,can be difficult to interpret accurately. Results of array analysesindicate that it not necessary to monitor the expression of all possiblegenes to classify the tumor accurately [5;9]. In fact, as exemplified byfindings made from studies of colon carcinomas, a majority of which havea preponderance of mutations of the APC and p53 genes [10], it appearsthat analysis of relatively few gene products would be adequate toclassify tumors. The types of genes to be monitored can be determined bytaking advantage of information that is usually known at the time ofsurgery, such as the location of the tumor (i.e., mammary gland,prostate, colon, lung, brain, etc.). The technology described herepermits one to measure the expression of several gene products in singlecells of frozen sections that are routinely prepared during surgicalprocedures. By focusing on genes whose expression has been found inmicroarray and other analyses to be most characteristic of a given tumortype, it will be possible to classify the tumor accurately. The devicestaught here permit this information to be determined in a rapid fashionand can be used to form the basis of instant decisions needed forpatient care.

The cells in a cancer have altered properties that enable them to evadeapoptotic mechanisms that normally limit cell growth. Some of theseinclude checks on the integrity of their genome and, when these are lostor become non-functional, cancer cells tend to accumulate mutations thatmake them more aggressive. Since not all the cells of a tumor have thesame mutations, the tumor can be heterogeneous. The heterogeneity ofsome tumors may even be due to the fact that they have originated fromseveral different cells, not just a single cell. Thus, to classify thetumor accurately, it is best to assess gene products from individualcells so that the degree of heterogeneity can be ascertained. It is alsoimportant to detect the existence and location of even a small number ofcells that have reduced sensitivity to natural regulatory mechanisms.The ability to do so would enable pathologists and surgeons to learn ifthe tumor contains cells that have characteristics indicative of a moreadvanced stage of cancer as well as to learn where they are within thetumor. If this information were available at the time of surgery, itwould enable the surgeon to tailor the surgical procedure appropriatelyfor each patient. For example, the absence of these cells might indicatethat it would not be essential to remove nearby or distant lymph nodesthat are not part of the tumor. In contrast, the presence of a fewadvanced cells in a small otherwise unremarkable tumor might be groundsfor more extensive surgery. Thus, it would be desirable to have a sensorthat could quantify gene expression rapidly in single cells of frozensections obtained at the time of surgery. Furthermore, this informationshould also affect the choice of post-surgical treatment such aschemotherapy and/or radiation therapy.

The therapeutic benefits of identifying cells that have alteredgenotypes and/or phenotypes that lead to pathological states have beenrecognized for many years. The need to classify these cells has led todevelopments of several methods for examining cells that range fromsimple staining procedures to highly refined approaches for identifyingspecific genes and gene products within the cell. Increased knowledge ofcell function offers a greatly expanded number of markers that can beused to assess the pathological status of single cells.

Several methods have been developed to study gene function in individualcells. Fluorescence Activated Cell Sorting (FACS) methods have permittedindividual cells to be isolated from complex cellular mixtures based onthe use of antibodies to a single surface protein. This method requiresdisrupting tissues into their component cells, which is a time-consumingprocess that makes FACS analysis poorly suited for use as a routinesurgical procedure. Techniques such as Fluorescent in situ Hybridization(FISH) are sufficient to detect single genes within cells of a tissue.The most sensitive of these techniques require considerable tissuepreparation, however, and are not sufficiently rapid for routine useduring surgery. Furthermore, the intrinsic fluorescence in cells andother factors often contribute to high background. This makes itessential to perform several time-consuming internal controls withoutwhich it would be impossible to interpret the analysis. Other propertiesof fluorescence, such as the ability of adjacent fluorophores tointeract with one another, a process known as Fluorescent ResonantEnergy Transfer (FRET), have been used to facilitate analyses of geneexpression. For example it has been found that fluorescentoligonucleotides can be used to detect mRNA products of single genescells based on the abilities of the oligonucleotides to bind to adjacentportions of the mRNA [11]. Nonetheless, these techniques can be plaguedby the high intrinsic fluorescence of cells. While it is possible tocircumvent this problem using time-resolved methods [12], this increasesthe complexity of the method substantially at the expense of assaysensitivity. In addition, there is a need to get the fluorophores intothe cells where they can interact with the mRNA. Thus, this approach isnot practical for routine examination of tissue sections. Efforts havealso been made to monitor gene products using fiber optic techniques[13]. These methods are also not applicable to tissue sections andsuffer from a very slow response time.

In summary, knowledge of the gene products that are associated withdifferent pathologies is accumulating rapidly. The public availabilityof the sequence of the human genome and advances in microarraytechnology has permitted the simultaneous semi-quantitative measurementsof large numbers of gene products. Array procedures have been used tocharacterize changes in gene expression in several types of normal andabnormal tissues. Indeed, comparisons of gene expression patterns intumor tissues with tumor recurrence and long-term survival of patientsfollowing surgery, chemotherapy, and/or radiation have enabledpredictions about the types of therapies that are most likely to bebeneficial [4]. As noted earlier, array procedures are not readilyadapted to analyses of single cells. Consequently, the data generated byapplication of this technique are confounded by the presence of analytesin non-tumor cells as well as by the fact that many tumors containdifferent types of abnormal cells. This makes it difficult to associategene expression with particular cells in even a semi-quantitativefashion. Furthermore, array analysis is time-consuming and not suitedfor the rapid estimation of gene expression while the patient is in theoperating room. Measurements of gene expression in single cells withinthe tumor would be of considerable value for classifying the tumor, akey component used to make informed decisions about the extent ofsurgery and subsequent therapies. It would also be applicable duringresearch to learn which gene expression products are most likely to havepredictive value. Finally, it would also be useful for studies of cellfunction during complex processes such as those that occur duringdevelopment and cellular differentiation.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate an overview of the sensor apparatus showing thesensor from three different perspectives. FIG. 1A shows an end view ofthe sensor.

FIG. 1B shows a top view of the sensor. FIG. 1C shows a side view of thesensor.

FIG. 2 shows the molecular beacon for β-actin.

FIG. 3 shows the steps in the preparation of biotinylated sensorsurfaces.

FIGS. 4A-4B illustrate the polarization routines. FIG. 4A showsnegatively charged oligonucleotides migrating towards the positivelycharged sensor surface. FIG. 4B shows the use of a waveform to preventpremature separation of the analyte and the detection reagent (i.e.,fluorescent PNA designed to contain a single positive charge).

FIGS. 5A-5B illustrate the principle of sensor operation in Example 2.FIG. 5A shows formation of the complex. FIG. 5B shows that during theseparation phase, the fluorescent complex migrates to the anode where itwould be observed and the fluorescent unbound PNA migrates to thecathode.

FIGS. 6A-6B illustrate TIRF illuminator for multiple objectives. FIG. 6Ashows a side view with the position of the light source and objective.FIG. 6B illustrates the manner in which the illuminator would be mountedon a microscope.

FIGS. 7A-7B illustrate a modification of the sensor that can be used forheating. FIG. 7A is an end view of the sensor and FIG. 7B is a side viewof the sensor.

FIG. 8 illustrates a microtiter well plate design.

FIGS. 9A-9F illustrate a polymer-based sensor device. FIG. 9Aillustrates the overall design of the polymer-based device, which isshown in an expanded schematic form. FIG. 9B illustrates the device asit is being assembled. FIG. 9C illustrates the device as it is beingused during electrophoresis. FIG. 9D illustrates the construction of theanode (component #1 plus component #2) and cathode (component #8 pluscomponent #9). FIG. 9E illustrates the construction of the anode andcathode assemblies. FIG. 9F illustrates the mounting of the “exposed”sensor sandwich on the camera.

FIG. 10A-B illustrates the migration of PNA labeled with a fluorophore(PNA*). FIG. 10A illustrates the migration of PNA labeled with afluorophore (PNA*) when it is free and bound to RNA in the sensorapparatus. FIG. 10B illustrates the migration of a fluorescent chargeddetection agent before and after its charges have been removed by anenzyme or a reaction with materials in or released from the tissuesection.

FIG. 11 illustrates design considerations for component #3.

FIGS. 12A-12D illustrate the illumination of the system. FIG. 12Aillustrates the arrangement of the system used to illuminate component#3 (or component #7, when used). FIG. 12B illustrates the illuminationused to distinguish colors. FIG. 12C illustrates a preferred type offilter that can be used in the device to permit distinguishing coloredfluorophores, if it is necessary to reduce the amount of scatteredlight. FIG. 12D illustrates a preferred mode for illuminating thesample.

SUMMARY OF THE INVENTION

The present invention provides a sensor device for detecting an analytein a sample in which an analyte is bound to a detection reagent to forma bound complex, wherein the device comprises:

(a) a sample (5) comprising an ionic analyte and a detection reagent ina conductive fluid, wherein the detection reagent has a net chargedifferent from the analyte;

(b) a first permeable polymeric hydrogel plate (3) and a first spacerplate (8), which plates provide a compartment for the sample;

(c) an anode (1) juxtaposed to the outside of the first hydrogel plateand not in contact with the sample;

(d) a cathode (9) juxtaposed to the outside of the first spacer plateand not in contact with the sample;

(e) a voltage generator (10) to apply an electric potential to the anodeand cathode; and

(f) a detector (11);

wherein the bound complex formed from the analyte and detection reagentis detected by the detector because the bound complex has a charge thatcauses it to migrate in a direction opposite from that of the unboundanalyte when the electric potential is applied.

The present invention also provides a method for detecting an ionicanalyte in a sample in which an analyte is bound to a detection reagentto form a bound complex, comprising the steps of:

-   -   (A) providing a sensor device comprising:

(a) a sample (5) comprising an ionic analyte and a detection reagent ina conductive fluid, wherein the detection reagent has a net chargedifferent from the analyte;

(b) a first permeable polymeric hydrogel plate (3) and a first spacerplate (8), which plates provide a compartment for the sample;

(c) an anode (1) juxtaposed to the outside of the first hydrogel plateand not in contact with the sample;

(d) a cathode (9) juxtaposed to the outside of the first spacer plateand not in contact with the sample;

(e) a voltage generator (10) to apply an electric potential to the anodeand cathode; and

(f) a detector (11); and

-   -   (B) adding the ionic analyte and detection reagent in the        conductive fluid to the compartment;    -   (C) applying an electrical potential via the voltage generator;        and    -   (D) detecting via the detector the bound complex formed from the        analyte because the bound complex has a charge that causes it to        migrate in a direction opposite from that of the unbound analyte        when the electric potential is applied.

The present invention also provides a sensor device for detecting andquantifying a gene product in a cell or tissue section sample byemploying an analysis reagent that binds to the gene product to form adetectable product comprising:

(a) a first and second coated plate, wherein the plates are parallel toeach other and are coated with a conductive material;

(b) a first and second conductive plate, wherein the plates are parallelto each other and are juxtaposed over the coated plates of (a);

(c) a first conducting tape connecting a first end of the coated platesof (a) and the conductive plates of (b) and a second conducting tapeconnecting a second end of the coated plates of (a) and the conductiveplates of (b);

(d) a first gasket insulator insulating a first end of the coated platesof (a) and the conductive plates of (b) and a second gasket insulatorinsulating a second end of the coated plates of (a) and the conductiveplates of (b);

(e) a voltage generator connected to the first and second conductiveplates to apply an electric potential to the conductive plates; and

(f) a detector;

wherein the first and second coated plates provide a compartment for acell or tissue section sample and a conductive fluid and an analysisreagent is provided in the sample or tethered to a surface of the firstor second coated plate such that when the voltage generator applies anelectric potential to the conductive plates, the detector will detectthe interaction between charged materials within the cell or tissuesection sample, migrating towards either surface of the coated plate,and the analysis reagent.

The sensor device may further comprise a heating means to heat thesample prior to, or during, detection of the sample and may furthercomprise a cooling means to cool the sample prior to, or during,detection of the sample. The detector may be a fluorescence,luminescence, colorimetry, or total internal reflection illuminationdetector or may detect by phase contrast microscopy, bright fieldmicroscopy, darkfield microscopy, differential interference contrastmicroscopy, confocal microscopy, or epifluorescence microscopy. Theelectrical potential may be applied perpendicular to the coated plateand may be constant or varied such that the overall effect is to haveeach plate have a net charge, such that charged analytes in the tissueswill migrate to one plate. The electrical potential may also be appliedperpendicular to the coated plate and may be alternated such that thereis no net charge on either plate, such that charged analytes willoscillate back and forth in the central space away from either platewhere they interact with analysis reagents.

The present invention also provides a method for detecting andquantifying a gene product in a cell or tissue section sample byemploying an analysis reagent that binds to the gene product to form adetectable product, wherein the analysis reagent is tethered to asurface of a sensor device, comprising the steps of:

-   -   (A) providing a sensor device comprising:

(a) a first and second coated plate, wherein the plates are parallel toeach other and are coated with a conductive material, and an analysisreagent is tethered to a surface of the first or second coated plate;

(b) a first and second conductive plate, wherein the plates are parallelto each other and are juxtaposed over the coated plates of (a);

(c) a first conducting tape connecting a first end of the coated platesof (a) and the conductive plates of (b) and a second conducting tapeconnecting a second end of the coated plates of (a) and the conductiveplates of (b);

(d) a first gasket insulator insulating a first end of the coated platesof (a) and the conductive plates of (b) and a second gasket insulatorinsulating a second end of the coated plates of (a) and the conductiveplates of (b);

(e) a voltage generator connected to the first and second conductiveplates to apply an electric potential to the conductive plates; and

(f) a detector; and

-   -   (B) adding a cell or tissue section sample and a conductive        fluid to a compartment within the first and second coated plates        of the sensor device;    -   (C) applying an electrical potential via the voltage generator        to the conductive plates;    -   (D) detecting via the detector the interaction between charged        materials within the cell or tissue section sample, migrating        towards either surface of the coated plate, and the analysis        reagent.

The present invention further provides a method for detecting andquantifying a gene product in a cell or tissue section sample byemploying an analysis reagent that binds to the gene product to form adetectable product, wherein the analysis reagent is soluble in thesample, comprising the steps of:

-   -   (A) providing a sensor device comprising:

(a) a first and second coated plate, wherein the plates are parallel toeach other and are coated with a conductive material;

(b) a first and second conductive plate, wherein the plates are parallelto each other and are juxtaposed over the coated plates of (a);

(c) a first conducting tape connecting a first end of the coated platesof (a) and the conductive plates of (b) and a second conducting tapeconnecting a second end of the coated plates of (a) and the conductiveplates of (b);

(d) a first gasket insulator insulating a first end of the coated platesof (a) and the conductive plates of (b) and a second gasket insulatorinsulating a second end of the coated plates of (a) and the conductiveplates of (b);

(e) a voltage generator connected to the first and second conductiveplates to apply an electric potential to the conductive plates; and

(f) a detector; and

-   -   (B) adding a cell or tissue section sample, a conductive fluid,        and a soluble analysis reagent to a compartment within the first        and second coated plates of the sensor device;    -   (C) applying an electrical potential via the voltage generator        to the conductive plates;    -   (D) detecting via the detector the interaction between charged        materials within the cell or tissue section sample, migrating        towards either surface of the coated plate, and the analysis        reagent.

The detector may be a fluorescence, luminescence, colorimetry, or totalinternal reflection illumination detector or may detect by phasecontrast microscopy, bright field microscopy, darkfield microscopy,differential interference contrast microscopy, confocal microscopy, orepifluorescence microscopy. The electrical potential may be appliedperpendicular to the coated plate and may be constant or varied suchthat the overall effect is to have each plate have a net charge, suchthat charged analytes in the tissues will migrate to one plate. Theelectrical potential may also be applied perpendicular to the coatedplate and may be alternated such that there is no net charge on eitherplate, such that charged analytes will oscillate back and forth in thecentral space away from either plate where they interact with analysisreagents. The gene products may be nucleic acids or proteins. Theanalysis reagent may be a biotin-streptavidin conjugate or may be amolecular beacon. Preferably, a mixture of molecular beacons labeledwith the same fluorophore is employed to detect a mixture of geneproducts associated with a tumor class. A second molecular beacon may beemployed as an internal control. Preferably, a first molecular beacon isemployed to detect a control gene product and a second molecular beaconis employed to detect a gene product of experimental or diagnosticinterest, wherein the first and second molecular beacons are eachlabeled with a different fluorophore that emits at a differentwavelength so that the first and second molecular beacons can besimultaneously analyzed. The control gene product may be β-actin. Thetransparent plates may be coated with indium tin oxide or tin dioxide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a rapid, sensitive, and accurate methodthat can be used to measure nearly any analyte. In particular, themethod can be employed to visualize the relationship between geneexpression and tissue morphology. The method utilizes an electricalpotential to promote the movement of the analyte from one site toanother causing the analyte to be concentrated in the region where themeasurement can be made. By controlling the electrical potential it ispossible to concentrate materials from tissue samples, electrophoresisgels, or any other media at a sensor surface and thereby enhance thesensitivity and the speed with which measurements can be made.Furthermore, the electrical potential can be used to reduce non-specificinteractions that occur during analysis and thereby facilitatemeasurement accuracy. The electrical potential can also be used to alterthe chemistry of the analyte and the sensor surface, and to immobilizesensor molecules at the surface via covalent bonds, coordination orphysical adsorption. Analysis occurs by the specific interaction betweenthe material that has migrated towards the surface of the plate andreagents that are attached to the plate or that are held near the platesurface. Because this analysis does not alter the relative positions ofcells or other factors that are being analyzed, it permits theidentification of analytes that are associated with specific cell typesor with specific portions of the material being analyzed. The sample mayalso be reduced or oxidized to increase the specificity and accuracy ofthe device. The method permits decisions to be made by physicians andpathologists at the time of the procedure and facilitates analysis bypersons less skilled in these tasks, such as technicians who do thepreliminary reading of Pap tests and other analyses that are preformedin high volume on a routine basis. The information will also be usefulfor making decisions regarding treatments after the procedures arecompleted.

In one embodiment, the present invention can be used to measure geneexpression products in tissue sections. These gene products can benucleic acids, such as messenger and other RNAs, or proteins such asenzymes and transcription factors. The method proposed for use withtissue sections involves placing the tissue sections or cells, includingthose taken at time of surgery, between two transparent plates or slidesthat have been coated with a material that conducts electricity or thatcan be made to conduct electricity. When an electric potential is placedon either side of the tissue, charged materials within the tissue can bemade to migrate towards either plate. Those with a net positive chargewill migrate towards the cathode and those with a net negative chargewill migrate towards the anode. The electrical potential on thetransparent plates, which serve as electrodes, can be constant or variedin a variety of fashions. When the potential is constant or when it isvaried such that the overall effect is to have each plate have a netcharge, charged analytes in the tissues will migrate to one electrode.When the potential is alternated such that there is no net charge oneither plate, charged analytes will oscillate back and forth in thecentral space away from either electrode where they interact withdetection reagents.

The method is not limited to tissue sections but can be applied todetect other agents and may not require the use of two slides.Metabolites that are altered as the result of changes in gene expressionmay also be detected.

In a second embodiment, the sample is recognized by a binding agent inan interaction that occurs in solution and that can take place at eithersurface of the sensor device, in the vicinity of either surface, or awayfrom either surface. The complex that is formed has a differentelectrical charge than the binding agent. Application of an electricalpotential across the plates results in the migration of the complextowards one of the plates where it can be measured. Since the bindingagent and the complex have different charges, it is possible to separatethe binding agent from the complex, a phenomenon that can be employed toreduce measurement noise. When operated in this fashion, the device canbe used to monitor any interaction that leads to a change in charge.This includes enzyme reactions in which enzymatic activity leads to achange in the charge of the substrate.

The actual measurement will be made when the charged materials reach thesurfaces of one or both plates. In most cases, the measurement willdepend on a change in fluorescence. There are two basic methods ofexamining fluorescence. In one method, a fluorophore will be attached tothe surface. Migration of the analyte to the surface will cause anincrease in fluorescence or a decrease in fluorescence of the bounddetection reagent. For example, binding of the analyte to a molecularbeacon would increase its fluorescence. While one could take advantageof a decrease in fluorescence caused by quenching, energy transfer, oreven destruction of the surface fluorophore (e.g., by proteolysis ornuclease digestion), this would be less sensitive due to the fact thatit would have a high background. The second method of detection dependson the ability of the analyte to cause the migration of a fluorophore tothe surface. In this case, the fluorophore detection reagent is eitheruncharged or charged in a way that would cause it to migrate to the sideof the device that is not being examined. Binding of the analyte to thefluorophore would change its net charge and cause it to migrate to thesurface that is being examined. Since the charge on a fluorophore couldalso be changed by cutting the fluorophore or modifying it (i.e., addinga phosphate), this procedure would also permit detection of enzymes.This method could readily be used with quantum dots, fluorophores thatare nearly indestructible and that are very bright.

Both methods have their advantages. The second method is preferablebecause it does not require surface labeling (a task that can requiredifficult chemistry), it enables the use of much higher reagentconcentrations of reagents, and it can produce very low backgroundbecause of the physical separation of the materials that occurs afterelectrophoresis. Advantages of the first method include the fact thatthe bound and free analytes are not separated, permitting detection oflower affinity interactions, and it can be used with a larger number ofoptical techniques. Indeed, since the fluorophore is attached to thesurface, there is no need to use optical techniques that limitillumination to the surface.

The sensor device can also be heated and/or cooled to facilitateinteractions between the reagents or even amplification of the analyte(i.e., by PCR). Fluorescence on the surface may be monitored using TotalInternal Reflection Methods (TIRF), including TIRF microscopy (TIRFM)using methods that are well known in the art. A lens-based method hasalso been devised for extending these measurements. Another procedurefor monitoring surface fluorescence involves the use of two photonmethods. In these methods, photons that have insufficient energy toexcite the sample individually are directed at the surface at the sametime. When the photons reach the surface, the sum of their energies willexcite the sample, enabling it to be detected. Another procedure thatcan be used is the employment of a lens that has a shallow depth offield that can be focused on the surface. Colorimetric methods can bealso used, i.e., when the analyte-detection complex reaches the surface,it causes the appearance of a color.

When tissue sections are to be examined, it will be useful to have amethod that can be used to scan the tissue sections automatically,freeing the surgeon or pathologist from spending time finding regions ofgreatest interest. Once these are detected by their fluorescence, theycan be examined manually.

As set out above, detection of the interactions between the analytes andreagents may be carried out using fluorescence techniques although othervisual methods including colorimetry and luminescence can be applied aswell. One of the most useful techniques for detecting nucleic acid geneexpression products such as mRNA employs molecular beacons. These can beattached to the surface of the sensor plate using a variety of methods.One of the most convenient involves attaching biotinylated molecularbeacons to surfaces that have been coated with streptavidin. In thismethod, the beacon is synthesized as a biotin derivative by standardmethods such as those employed by companies specializing in molecularbeacon synthesis including IDT Technologies, Inc., Coralville, Iowa52241, USA. Attachment of the biotinylated molecular beacon to thesurface of the plate can be performed by attaching it to streptavidinthat has been attached to the surface of the plate. Attachment ofstreptavidin to surfaces is well known in the art and can accomplishedby reacting it with biotin derivatives that are covalently attached tothe plate or by permitting it to interact with bovine serumalbumin-biotin conjugates such as those obtained from Sigma ChemicalCo., St. Louis, Mo. 63195, USA that have been adsorbed to the platesurface. Introduction of a charge between the plates of the sensordevice promotes migration of the mRNA from the tissue to the positivelycharged surface of the sensor. This can be facilitated by theintroduction of small quantities (i.e., 0.1-2%) of non-ionic detergentssuch as octylglucoside, which disrupt the plasma membranes that surroundthe cells in the tissue sections. It can also be facilitated by varyingthe charge on the plate surface in a fashion that prevents thenegatively charged nucleic acid from sticking directly to the platesurface. Interaction of the mRNA gene products with the molecularbeacon, a process that can be made to be highly specific by design ofthe molecular beacon using methods that are standard in the art willlead to increased fluorescence. Since this will be immediately above orbelow the material being analyzed, the amount of fluorescence will beroughly proportional to the amount of nucleic acid within cells or otherlocal portions of the material being tested.

It is not necessary to use fluorescent reagents that are covalentlyattached to the surface of the sensor for analysis. For example, mRNAcan be monitored using peptide nucleic acids (PNA), which are analogs ofnucleic acids that have the sugar-phosphate backbone replaced by peptidebonds. PNA have the same binding specificity as nucleic acids and can bedesigned using the same principles as are well known in the art toconstruct oligonucleotides that interact with nucleic acids. PNA aresuperior to nucleic acids for measurement in the sensor, however,because they lack the strong negatively charged phosphate backbonestructures characteristic of nucleic acids. Thus, PNA are essentiallyneutral in physiological buffers and do not have a great propensity tomigrate to either surface of the measuring device. When they bind tomRNA or other nucleic acids, the complex becomes negatively charged dueto the negatively charged backbones of the part of the complex derivedfrom the nucleic acid. Thus, the complex will migrate towards an anode.If the PNA are made to contain a fluorophore, formation of the complexwill cause the fluorophore to migrate towards the anode where it can bereadily detected using TIRFM, confocal microscopy, microscopictechniques that employ two or three photons to excite the sample, or byuse of an objective that has a very shallow depth of field. If thefluorophore that is attached to the PNA is positively charged, unboundPNA molecules will migrate towards the cathode. Thus, by measuringfluorescence at the anode, it is possible to detect and quantifyspecific mRNA gene products in samples.

While nearly any procedure capable of detecting fluorescence can be usedto detect the material, it is often most useful to perform the techniquein an optical microscope. In cases where the background fluorescencethat may be present in tissues and tissue sections is found to limit thesensitivity of the technique, one can also apply microscopic techniquessuch as TIRFM a device that is constructed specifically for this purposeand that is readily adapted to routine use. TIRFM is a very sensitiveprocedure that permits studies of single molecules and has even beenused to investigate the folding of single molecules of RNA [14]. TIRFMtakes advantage of a physical characteristic of electromagneticradiation that occurs when light reflects from the interface between twooptical media that differ in refractive index. In TIRFM a beam of lightis passed through a material of high refractive index (e.g., glass,fused silica, sapphire) such that it reaches an interface with amaterial of lower refractive index (e.g. aqueous solution, tissuesection). When the angle of incidence is below a value known as thecritical angle, all the light is reflected back into the material ofhigh refractive index. A standing electromagnetic or “evanescent” wavewill be generated at the interface. Its energy will be maximal at theinterface and will decay exponentially as a function of distance fromthe surface of higher refractive index, e.g., as the electromagneticwave penetrates into an aqueous medium. The energy in the evanescentwave light can excite fluorophores that are attached to the surface orthat are in close proximity (100-400 nm) to the surface of highrefractive index. The limited distance traveled by the evanescent waveis responsible for the ability of TIRFM to illuminate material that ison or very near the surface of the TIRFM sensor (i.e., the material ofhigh refractive index). As a consequence of the physical principle thatunderlies TIRFM, the unwanted background light that results from theintrinsic fluorescence of tissue samples that is often a problem forother types of fluorescent microscopy is virtually eliminated. This highsignal-to-noise ratio is responsible for the ability of TIRFM to detectand quantify trace amounts of material in the face of an overwhelmingamount of non-specific contaminating debris.

Use of TIRFM can also permit use of the sensor for analysis underconditions in which the reagents that are being used to detect theanalyte are not necessarily attached to the sensor surface. Thus, whenfluorescent PNA are added to the tissue sections that have been treatedwith agents such as non-ionic detergents that disrupt the integrity ofthe cell membrane but not the overall architecture of the tissue, theywill interact with nucleic acid gene products (i.e., mRNA and other RNApolymerase derived nucleic acids). Application of an electric potentialwill cause the fluorescent PNA-RNA hybrid complexes to migrate to thesensor surface where they can be detected. Since multiple PNA can beemployed and since multiple fluorophores can be employed, this techniquepermits simultaneous measurement of many different analytes, asignificant advantage during studies to identify gene expressionproducts.

Addition of an electrochemical polarization to the sensor surface usedin TIRFM can increase the sensitivity and the speed of analysis further.Coating of the TIRFM sensor chip with a thin layer of indium tin oxide(ITO), tin dioxide (SnO₂), or several other metals does not affect itsability to be used for TIRFM at near ultraviolet or visible lightwavelengths. Application of an electrical potential to the metal coatingcan be used to enhance the concentration of material at the sensorsurface. This can increase the sensitivity of detection as well as thespeed with which the measurements can be made. For example, by varyingthe electrical field on the TIRFM sensor surface, it is possible tofacilitate the migration of nucleic acid oligomers to the surface of thesensor where they can hybridize with others that are on the sensorsurface. The presence of an electric field can also facilitate therelease of mRNA from tissue sections by disrupting the plasma membranes,a process known as electroporation. This will enhance the migration ofmRNA towards the anode sensor surface. It will also facilitateinteractions of mRNA with other agents such as PNA. When appropriatefluorophores such as molecular beacons are attached to the sensorsurface, it is possible to use this principle to selectively measurenearly any gene product in single cells. Since tissue sections areapplied directly on the sensor surface during surgery, this procedureresults in a rapid and quantitative analysis of gene products withincells and will permit distinguishing the expression patterns cellswithin the tissue.

Several different types of fluorophores have been incorporated intomolecules than can be used for detection and companies such as MolecularProbes, Eugene, Oreg. and Integrated DNA Technologies (IDT), Coralville,Iowa market them. One of the most useful properties of fluorophores istheir ability to undergo resonance energy transfer (RET), also known asfluorescent resonance energy transfer (FRET). RET between adjacentfluorophores occurs when the adsorption spectrum of one overlaps thefluorescence spectrum of the other. According to principles firstestablished by Förster [15], the amount of RET between two fluorophoresvaries as the inverse of the distance between them to the sixth power.Thus, RET will be nearly quantitative when the fluorophores are adjacentand virtually undetectable when the fluorophores are separated by aslittle as 100 Å and, in many cases, even less. During RET, energy fromthe fluorophore that adsorbs light at shorter wavelengths is transferredto that of the fluorophore whose adsorption spectrum overlaps theemission spectrum of the first fluorophore. This leads to a reduction inthe amount of light emitted from the first fluorophore and an increasein the amount of light emitted from the second fluorophore. Thereduction of light emitted by the first fluorophore can be used toestimate the distance between the fluorophores. It can also be used toassess the formation of a complex between two molecules that are labeledwith fluorophores that are capable of undergoing RET. RET between twofluorophores usually leads to a change in the spectrum of light that isemitted. Measurements of the emission spectrum are also useful forquantifying the distance between the two fluorophores and have beenwidely used to monitor enzyme reactions, such as that seen in thepresence of β-lactamase. RET is also useful for quantifying analytes aswell as interactions between ligands and receptors. Its uses for thesepurposes are well known.

Not all molecules that adsorb light fluoresce. When RET occurs between afluorophore and non-fluorescent molecule, the latter will quench thefluorescence of the fluorophore. When the fluorophore and the quenchingmolecule are sufficiently close to one another, all or nearly all thefluorescent energy will be quenched and little or no light will beemitted. This property is particularly useful for detecting analytesthat disrupt contacts between the fluorophore and the quenching moleculesince the amount of light that is emitted will be directly proportionalto the amount of the analyte. In the absence of analyte, none of thelight will be emitted, resulting in a very low assay blank. Thisproperty led to the development of “molecular beacons” [16], hairpinshaped molecules designed for the measurement of nucleic acids. In theabsence of analyte, the end of the molecular beacon that contains thefluorophore is held adjacent to the end of the molecular beacon thatcontains the quenching molecule by hydrogen bonds similar to thoseresponsible for the hybridization of nucleic acids. When theseinteractions are disrupted by the binding of a second molecule ofnucleic acid, the distance between the fluorophore and the quenchingmolecule exceeds that needed for RET and the fluorescence becomesreadily visible. By combining RET and TIRFM, it is possible to enhancethe desirable properties associated with each technology, therebyfacilitating the measurements of analytes. The combined sensitivity ofRET and TIRFM has permitted studies of single molecules [14].

In a preferred application of the device, the application of an electricfield causes the analyte to migrate to the sensor surface where itinteracts with an immobilized molecular beacon or other fluorophore.This results in a change in fluorescence of the immobilized fluorophore.Molecular beacons are particularly well suited for use in this devicesince their fluorescence increases upon interaction with nucleic acidsin a highly sensitive and predictable fashion. One of the limitations ofthis type of sensor is the need to attach the agent to the sensorsurface. This requires additional steps in sensor construction and canbe limited by the amount of material that can be attached to thesurface. While these limitations are usually not severe, they canincrease the costs of sensor construction. A wide range of chemistriesis available for attaching materials to the surface of sensors used inthe device and reagents for doing so are available from severalcompanies including United Chemical Technologies, Inc., 2731 BartramRoad, Bristol Pa. 19007. Furthermore, it is possible to increase the“depth” of the surface considerably by attaching compounds such asdextran that can serve as additional attachment points.

It is not necessary to attach the detection reagent to the surface tooperate the device, however, and another preferred embodiment of thesensor is based on the use of soluble detection reagents. These can haveconsiderable advantages to the use of surface bound material. First,since soluble reagents are not coupled to the sensor surface, their usefacilitates sensor design by eliminating the surface-coupling step.Second, they can often be used in massive excess, a phenomenon that canincrease the sensitivity and speed of detection. Third, they can bedesigned in a manner that prevents them from reaching the surface unlessthey have interacted with the analyte. This can reduce the backgroundfluorescence observed in the absence of analyte. Indeed, the excessreagent can be designed such that it will migrate away from the sensorsurface during analysis, a phenomenon that can minimize the backgroundfurther. Fourth, interaction of the detection reagents and the analytecan take place away from the surface, which minimizes artifacts causedby surface phenomena. These include non-specific adsorption to thesurface, which can prevent interactions between the analyte and thedetection reagent. While these can also be minimized by varying thepotential on the surface of the device, this adds an additionalcomplication to the analytical procedure. Fifth, these reagents arereadily adapted to use with quantum nanodots, fluorophores that are notreadily photobleached and that have a very high quantum efficiency.Quantum nanodots can be purchased from the Quantum Dot Corporation,26118 Research Road, Hayward Calif. 94545, USA. Furthermore, quantumnanodots can be excited at short wavelengths and have narrowfluorescence spectra. This permits the simultaneous detection ofmultiple analytes following excitation with only a single laser beam, amajor advantage in analysis of gene expression where it is desirable toobserve many gene products at one time.

The need for analytes to reach the sensor surface before they can beobserved, a property of TIRFM that facilitates distinguishing specificfrom non-specific interactions, can result in slow response times. Thiscan also reduce the sensitivity of TIRFM, particularly if the substanceto be measured is prevented from reaching the sensor surface. Geneexpression products such as mRNA or proteins that are held in tissuesections would not be expected to section such that charged analytes aredriven to the surface of the sensor where they can be detected. Theapplication of a charge perpendicular to the tissue section also reduceslateral diffusion of the gene products thereby increasing the likelihoodthat the fluorescence observed is associated with the cell that isexpressing the gene. In addition, by varying the charge, it is possibleto accelerate interactions between surface molecules and to reducenon-specific binding.

TIRF can also be monitored without the use of a high-magnificationmicroscope lens. In this case one loses the spatial resolution needed toidentify individual cells within a sample. Nonetheless, there are timeswhen it useful to monitor light emission over a large areas, such asduring efforts to scan the perimeter of a tumor to determine if theedges have been removed during surgery. There are few limits to the sizeof the TIRF sensor and it is envisioned that sensors of sizes other thanthose used commonly by pathologists will be of value for the technique.

Measurements of TIRFM can be done at several different magnificationsthrough the use of an objective prism. High magnification TIRFM usingcommercially available 60× and 100× microscope objectives can currentlybe accomplished using devices that have been specifically designed forthis purpose. Useful equipment for this purpose can be purchased fromNikon microscope dealers such as Micron Optics, 240 Cedar Knolls Road,Cedar Knolls, N.J. 07927 USA. In these devices, a laser beam is directedthrough the objective, an oil layer, and a thin coverslip ofapproximately 0.17 mm. These devices are excellent for visualizingfluorescence in tissue samples. When used with differential interferenceoptics (DIC), these microscopes can also be used to monitor the cellsfrom which the analytes are derived.

Due to the high power of the objective lenses that are used in thecommercial microscopes for TIRFM, it is difficult to scan tissuesections in a rapid fashion. There is a need for lower power TIRFM thatcan also be used with the sensor. As taught here, this is met bydesigning a new method for illuminating the samples. The use of thisstrategy to monitor a broad image permits much more rapid scanning ofthe sample.

Data collection can be made using a charge coupled device (CCD) cameraor related cameras of sufficient sensitivity, many of which areavailable commercially and are available from microscope dealers such asMicron Optics. Intensified CCD cameras are also available that are muchmore sensitive. These can also be purchased from most microscopedealers. Measurement of light intensity can also be done usingphotomultipliers that are attached to one of the optical ports on mosthigh quality microscopes. One useful instrument that has been designedfor this purpose can be purchased from C&L Instruments, 314 Scout Lane,Hummelstown, Pa. 17036 USA.

Even with the use of low power objectives, it is often desirable to scanthe surface of the sensor. This permits one to detect gene products insubsets of tissue sections and thereby distinguish normal andpathological tissues. This process can be accomplished manually bymoving the microscope stage that holds the sensor. It can also beaccomplished automatically using computer driven stages that areavailable from most microscope dealers. By combining the use of computerdriven stage movements and data collection, it is possible to devise animage of the entire sensor surface at high resolution. The operator canthen examine those regions of particular interest, a time saving featureof the method.

The analytical techniques taught here are not restricted to the analysisof nucleic acids, although this will be an important use. For example itis possible to measure proteases by permitting them to cleave specificsubstrates that are attached to the sensor surface. One such methodinvolves the preparation of peptides that contain a fluorophore and aquencher. Proteolysis of the peptide liberates the fluorophore from thequencher, resulting in enhanced fluorescence. Proteolysis can alsoremove charged components of the substrate that permits it and itsattached fluorophore to migrate to the sensor surface for observation.Similarly, the technique can be applied to the measurements of kinasesand phosphatases, enzymes that alter the phosphorylation status andhence the charge of an analyte. Changes in the charges of fluorescentkinase and phosphatase substrates can be used to promote migration ofthe substrates to a sensor surface where they can be measured. Thisforms the basis for the enzymatic analyses of these agents as well.

It is not essential to use fluorescent techniques for detection of theanalytes that are to be measured. Enzymatic analytes can be often bedetected by virtue of their enzymatic activity which can lead to thedeposition of colored reagents on the surface of the sensor.

As setout above, the present method can also be used to measure changesin the charge of any fluorescent material caused by interaction with ananalyte, including a binding molecule or an enzyme. It can also becaused by a cascade of events such as multiple enzyme-coupled reactions.

The present invention is further illustrated by the following examples,which are not intended to limit the effective scope of the claims. Allparts and percentages in the examples and throughout the specificationand claims are by weight of the final composition unless otherwisespecified.

EXAMPLES Example 1 A Sensor Device to Monitor Gene Expression in FrozenTissue Sections in which the Analysis Reagents are Tethered to OneSurface of the Device During the Entire Analytical Procedure

FIG. 1 illustrates the features of a sensor device that will enable themeasurement of gene products in cells of tissue sections. This preferredembodiment of the device consists of two plates placed on opposite sidesof the material to be analyzed (i.e., the tissue sections). While itwould be possible to detect some gene products by pressing the platesagainst the tissue sections, this is relatively inefficient process andis difficult to control adequately. A preferable mode of operation is tointroduce an electrical field between the plates perpendicular to thetissue as shown in FIG. 1. The potential used can be varied within widelimits but should usually be less than that which promotes theelectrolysis of water to prevent the accumulation of gas bubbles in thedevice. Thus, for frozen tissue sections that are roughly 200 μm thick,this will result in an electrical potential of 50 volts per cm more orless, a value that is much greater than the amount needed to promoterapid electrophoresis of nucleic acids such as mRNA. The electrophoreticmobility of the mRNA in tissue samples can be impeded by the cellmembranes, however, even when the tissues are partially damaged byfreezing and thawing during tissue sectioning. Gene products can usuallybe made more available for analysis by the inclusion of agents such asnon-ionic detergents (e.g., 0.1-1% octylglucoside) or other agents thatdisrupt cell membranes without drastically altering the cytoskeletal andother structural components of the cell. Disruption of the tissue can beminimized by using the smallest amounts of these agents possible. Careshould be taken to reduce tissue damage when histological analysis ofthe tissue sections is to be compared with the results of geneexpression analysis.

There are two principle methods that can be used to detect negativelycharged RNA polymerase generated gene products using the deviceillustrated in FIG. 1. In one, the detection reagent (e.g., a molecularbeacon) is attached to the surface of the plate that will serve as theanode. In the other, which will be described in Example 2, the detectionreagent becomes located near the anode during the procedure.

Attachment of detection reagents to the sensor surface can be done by avariety of methods. One of the most convenient is to use abiotin-streptavidin conjugation procedure. In this method a biotinmoiety is attached to the surface directly by chemically attaching abiotin derivative to a properly derivatized surface or indirectly byadsorbing a bovine serum albumin biotin complex to the sensor surface.The biotinylated surface is then reacted with streptavidin, a proteinthat contains four biotin binding sites. Binding of streptavidin to thesurface creates a biotin binding site on the surface, which can be usedto immobilize biotinylated detection reagents such as biotinylatedmolecular beacons. Incorporation of biotin into the beacons can be doneat the time they are synthesized. For example the beacon illustrated inFIG. 2, which was designed to recognize β-actin, contains a biotin thatwas incorporated during its synthesis by IDT DNA Technologies, Inc. Thiswas done to permit its attachment to streptavidin that was purchasedfrom Sigma, St. Louis, Mo., 63178, which had been attached tobiotinylated-bovine serum albumin (also purchased from Sigma) that hadbeen adsorbed to the surface of indium tin oxide (ITO) coated slidespurchased from Delta Technologies. USA (FIG. 3).

Many methods for preparation of chemically biotinylated ITO surfaces arewell known in the art. One method that is useful involves cleaning ITOcoated slides by treating them with H₂O/H₂O₂/NH₃ in a ratio of 10:2:0.6at 55° C. for 75 minutes followed by baking them in a vacuum oven at165° C. for 150 minutes to remove water. The slides are then cooled indry nitrogen and treated with 0.5% 3-aminopropyltrimethoxysilane intoluene. Both reagents can be obtained from Sigma-Aldrich, St. Louis,Mo. They are then washed with methanol and the resulting surface aminogroups are biotinylated by reacting the slides with a biotin analog thatis reactive with amino groups such as biotinamidocaproate,N-hydroxysuccinimidyl ester obtained from Molecular Probes, 29851 WillowCreek Road, Eugene, Oreg. 97402.

The chemically cleaned slides can also be treated with other agents thatpermit them to be derivatized with thiol, aldehyde, and other groupsthat facilitate conjugation with biotin containing and other compounds.They can also be treated with agents that cause them to be derivatizedwith polyethylene glycol (PEG) and PEG derivatives that can be purchasedfrom Shearwater Corp. (U.S.), 1112 Church Str., Huntsville, Ala. 35801.They can also be treated with reagents such as Sigmacote obtained fromSigma, that renders the surface hydrophobic and that facilitates theadsorption of biotinylated serum albumin.

Introduction of an electrical potential across the ITO or other metalcoated slides used to fabricate the optically transparent chamber wallswill cause negatively charged gene products such as mRNA to migratetowards the anode where they can interact with detection reagents suchas molecular beacons. Indeed, molecular beacons are preferred detectionreagents since they usually have low background fluorescence in theabsence of analyte and can be designed to interact specifically withpredetermined gene products using methods well known in the art. Indeed,companies that specialize in the synthesis of DNA and molecular beaconssuch as IDT DNA Technologies, Inc. offer a service in which they assistin the design of properly functioning beacons.

The molecular beacon will become much more fluorescent when it binds theanalyte for which it has been designed, a phenomenon that causes theshape of the beacon to be altered and that displaces the quenching agentfrom the fluorophore. For the mRNA to interact with the beacon, it musttravel from the cellular milieu to the anode sensor surface. This isfacilitated by the presence of the electric potential. Interaction ofthe mRNA with the molecular beacon can be enhanced by varying thepotential used to cause migration of the gene product to the anode. Adiagram representing a typical polarization pattern that can improve theinteraction of the mRNA and the beacon is illustrated in FIG. 4. Manyvariations on this theme will give adequate mRNA beacon interactionsthat are useful for measurement of gene expression, however, and it isnot essential to use that illustrated here. Variation in the potentialcan be performed with a potentiostat or similar device. Usefulinstruments include that from CH Instruments, 3700 Tennison Hill Drive,Austin, Tex. 78733, USA.

While a single molecular beacon can be used during analysis, it isusually preferable to employ at least two different beacons, one ofwhich is intended to serve as an internal methodological control. Thisbeacon can be made to detect gene products such as β-actin that arefound in abundant amounts in most cells and whose expression is notchanged significantly during most pathologies. The other beacon can bemade to detect products that are of experimental or diagnostic interestand should be labeled with a fluorophore that emits at a differentwavelength to permit its simultaneous analysis with the control beacon.The finding that the ratios of these gene products change providesstrong indication that significant changes in gene expression haveoccurred within the tissue. Furthermore, many tissue sections willcontain more than one cell type. Another control would be to compare theexpression of actin in each cell type with the expression of the geneproduct that is associated with a pathological condition.

The choice of the gene products to be measured for experimental ordiagnostic purposes will depend on the results of preliminary studies orof published microarray analyses, many of which are already known tothose familiar with the art. Furthermore, it may be desirable to monitormultiple gene products of diagnostic interest at the same time. Forexample, as noted earlier, microarray analysis has indicated thatseveral different gene products are associated with specific types ofbreast carcinomas. By using mixtures of beacons that are labeled withthe same fluorophore and that recognize several gene products associatedwith tumor class one can increase the chances of detecting this type oftumor. This is because the interaction of any or all of these geneproducts with these beacons will be associated with a particularfluorescent emission spectrum. By labeling pools of beacons thatrecognize gene products associated with a different type of tumor with afluorophore that has a different emission spectrum, it is possible todetect and classify pathological cells derived from more than one classwithin the tumor or to more accurately classify the tumor type, asignificant advance in diagnostic practice. Since analysis can be doneon sections obtained at the time of surgery, use of the sensor makes itpossible for the surgeon and pathologist to modify the surgicalprocedure in the most appropriate fashion for the patient during theprocedure.

There are two principle advantages that accrue from operating the sensorusing detection reagents that are attached to its surface. The first issimplicity of analysis. Since the detection reagents are physicallyseparated from the tissues throughout the procedure, it is not necessaryto use methods that limit fluorescence excitation to the anode orcathode. Thus, while procedures such as TIRFM and multiple photonexcitation can be used to monitor interactions between the beacons andthe gene products on one sensor surface, the fact that the beacons arefound only on this surface means that these techniques are not required.Indeed, it is often possible to use standard fluorescence microscopetechniques when the background illumination can be adequatelycontrolled. This reduces the costs of the instrumentation required. Andsecond, use of surface bound fluorophores does not require physicalseparation of bound and non-bound analytes. This permits monitoring oflow affinity interactions. While this is not a problem with themolecular beacons, it can be an issue for other types of analyticalprocedures such as interactions between fluorophores and surface boundproteins.

The advantages of using immobilized detection reagents can be offset byseveral factors including difficulties in attaching them to the surface,limits to the amount of material that can be attached to the surface,effects on ligand recognition caused by their attachment to the sensorsurface, the need to employ organic dyes that can photobleach, and theinfluence of non-specific interactions. The latter can often beminimized by the use of agents such as bovine serum albumin andpolyethylene glycol to block these interactions. The limitation on thenumber of groups that can be placed on the sensor surface can be offsetin part by increasing the surface area by coating it with dextran andother agents that serve as attachment sites. These techniques are allwell known to those familiar with the art.

Example 2 A Sensor Device to Monitor Gene Expression in Frozen TissueSections in which the Analysis Reagents Move with the Gene Products tothe Anode During Analysis

The second preferred embodiment, the device shown in FIG. 1, employsdetection reagents that are not attached stably to either sensorsurface. Analysis depends on the migration of the detection reagent toeither the cathode or anode following interaction with the analyte. Thisapproach circumvents many of the limitations that result from usingsurface immobilized detection reagents. Detection occurs when thecomplex reaches the one or other surface, depending on its charge.

A diagram outlining the mechanism by which this sensor operates is shownin FIG. 5. Basically, the agents that interact with the analyte areeither uncharged or weakly charged such that they tend to migrate to thesurface of the device opposite that being used to sense the analyte.mRNA gene products can be measured in this device using PNA (peptidenucleic acids), which are similar to ribonucleic acids except that theribose-phosphate backbone is replaced by a peptide bond. This makes themuncharged but does not affect their abilities to form heterodimers withcomplementary RNA sequences. These can be attached to fluorophores andit would be expected that they can also be attached to quantum nanodots.The latter reagents would have significant advantages due to theirresistance to photobleaching and their high intrinsic fluorescence.Binding of mRNA to the fluorescent PNA molecules causes them to becomenegatively charged, a phenomenon that causes them to migrate to theanode sensor surface where they can be detected by their fluorescence.

There are several advantages to detecting analytes using solublereagents that can be separated in an electric field. First, there is noneed to attach them covalently to the sensor surface. This simplifiesthe design of the device. Second the fluorophores migrate to the sensorsurface only when they have formed a complex with the analyte, aphenomenon that provides an intrinsic mechanism to limit backgroundfluorescence. In fact, since the PNA-fluorophore complex can be made tohave a weak positive charge, molecules that are not bound to the mRNAgene products will migrate away from the sensor surface. As a result, amassive reagent excess can be used within the device without causing anunacceptable increase in background noise. The fact that a larger amountof these reagents can be used in the device also increases itssensitivity and the speed with which it can be operated. Finally, aswill be noted in later examples, the mechanism that underlies thisanalytical approach can be used to monitor gene products other thannucleic acids.

These advantages of using soluble reagents for analysis of nucleotidebased gene products are offset in part by the requirement thatillumination be limited to the anode sensor surface. One practicalapproach for doing this is to use devices that illuminate the surface bytotal internal reflection. This limits illumination to the surface ofthe sensor used for detection. Equipment for TIRFM is commerciallyavailable from microscope dealers who handle instruments made by eitherNikon or Olympus. Instruments purchased from these companies are limitedto relatively high power objectives, however (i.e., 60× and 100×). Thiscan make it difficult to scan rapidly an entire sensor surface. Thereare other strategies for performing TIRFM that can be used with lowerpower objectives. These involve illuminating the sample through a prismsuch as that shown in FIG. 6.

Another means of illuminating the anode surface is to use two or threephoton microscopy or confocal microscopy. In the former approach, theanode surface would be illuminated such that that single photons areunable to excite the sample. Focusing the illumination source on thesensor surface to cause it to be illuminated “simultaneously” by two ormore photons provides sufficient energy to obtain fluorescence emission.The major limitation to the routine use of this type of illumination isits high cost.

Separation of the bound and free detection reagents is done byapplication of the electric field, which causes the bound detectionreagent to migrate to the anode when the complex is negatively chargedor to the cathode when the complex is positively charged. The rate atwhich the analyte will reach the surface will depend on the differencein potential between the plates, the frequency with which the potentialon the plates is changed, the size and charge of the analyte, andfactors that may limit its ability to migrate to the surface of theplate. Variations in the electric field can be very useful for causingthe complex to form. Thus, by alternating the electric field, one cancause charged analytes to migrate back and forth within the region ofthe sensors. This creates a mixing effect that can enhance interactionsbetween the analytes and the detection reagents that facilitateformation of the complexes.

Example 3 Details of Sensor Construction

The sensor described in FIG. 1 contains two glass, quartz, sapphire,mica, plastic, or other plates that are optically transparent at theillumination and fluorescent wavelengths to be used. This permits directvisualization of fluorescence or other optical events that result frominteractions of the analyte with materials in the sensor. It is oftenconvenient to use standard microscope slides or coverslips forconstruction of the optical portions of the sensor and it is notnecessary that both slides be made of the same material. In fact, unlessthe sensor is to be used for visual observation of its contents, it isnot necessary that both surfaces of the sensor be constructed ofoptically transparent materials. Indeed, it is possible to remove onesurface of the sensor prior to examining its contents.

The sensor surfaces are coated with ITO, SnO₂, or other conducting orsemi-conducting materials that are also optically transparent at thewavelengths to be used. This is done to enable an electric potential tobe developed between these two surfaces. While this is a preferablemeans of designing the electrical components of the sensor since itpermits both the optical and electrical components to be combined,workable sensors can be envisioned that would contain conducting gridsor membranes in place of one or both of these surfaces.

The device outlined in FIG. 1 contains a second metal coated surfacethat is transparent to light. It is not essential that this surface betransparent to light unless one wants observe the tissue sections byphase contrast or other regular light microscopic techniques withoutremoving it. In some cases, it may be desirable to remove the surfaceprior to observation by regular light microscopy since this will permitthe tissue to be stained using a histological dye before or after theanalysis by TIRFM. It is also not essential to use a solid surface asthe electrode. For example it is possible to use a metal screen, metalgrid, wire, semitransparent metal coating, or any other device that canbe used to apply a voltage across the tissue section.

Several methods can be used to deliver an electrical potential to thesurface of the plates. In one procedure, the entire plate is coated withITO or other conducting metal. When this is placed on a metal wire orother conducting surface, it will permit the introduction of anelectrical potential on all portions of the plate, including that incontact with the sample. Another method of connecting the conductingsurface of the plate to the wire or conducting surface must be used whenonly one surface of the plate that contacts the sample is coated withITO or conducting metal. Use of plates having only a single coatedsurface can facilitate the optical transmission of the device, aproperty that is often critical at ultraviolet or near ultravioletwavelengths. One means of making the appropriate electrical contactinvolves placing a wire directly on the metal surface of the plate. Thisapproach suffers from the difficulty of maintaining sufficient contactbetween the wire and metal coating on the surface to facilitate uniformelectrical conduction, particularly when the device is subjected torepeated handling. To circumvent this, one can glue the wire to themetal coated surface using material obtained from Delta TechnologiesLimited, 13960 North 47^(th) Street, Stillwater, Minn. 55082, USA.Alternatively, one can place a thin strip of metal on the conductingsurface of the plate. This can also be glued in place. A preferredmaterial for this can be purchased from Schlegel Systems, Inc.,Rochester, N.Y. 14623, USA. One thin strip that is particularly usefulis their Conductive Anti-Tarnish Copper Tape which comes in a variety ofwidths, contains one sticky surface, and is heat stable at 121° C.,making it autoclavable. This permits construction of sterile sensorsthat can be used as cell culture growth chambers. The resistance betweenthese tapes and that of the ITO surface of glass microscope slidespurchased from Delta Technologies is less than 1 ohm. FIG. 1 shows oneway that this strip can be located in the device. In this position, itpermits good electrical contact between the surface of the opticallycoated material and a brass holder. Since the coating is present only onthe ITO coated portion of the sensor, this portion of the sensor can bechanged easily. This feature is particularly desirable when the sensorsurface is to be produced in a fashion that makes it disposable. Using adoubly coated material permits the optical surface to be mounted tightlyto the holder, a particularly desirable feature when the entire deviceis to be disposable.

The need to prevent electrical contacts between the two plates of thedevice shown in FIG. 1 can be met by introduction of an insulatorbetween the two plates. It is often convenient to prepare this from aflexible material that permits a good seal such as a PDMS(polydimethylsiloxane) membrane or a silicone rubber gasket. This can beof nearly any thickness but it is preferable that it be similar inthickness to the sample being analyzed. The spacer can also consist ofshort posts and need not surround the sample as is shown in FIG. 1. Thespacer can also be molded into one or both surfaces during production.The composition of the spacer or gasket will depend on how the device isto be used. For most uses, it should be made of a non-reactiveinsulating rubbery material that makes a good seal with the surface ofthe sensor and prevents fluid leakage. The spacer can be glued to onesensor surface, if desired to obtain a better seal. This creates ashallow open chamber that facilitates addition of the conducting fluid,the next step in assembling the sensor sandwich.

Electrical contacts between the sensor and the sample occur through aconducting fluid. This can be nearly any dilute buffer that is capableof conducting electricity. The pH of the buffer should be chosen torender the analyte charged such that it migrates towards the surfacethat is to be observed. This includes the surface that coated(Example 1) or that to which the analyte-detection complex will migrate(Example 2). The type of buffer to be used in the connecting fluid willvary with the sample being analyzed. Analysis of RNA transcripts can beanalyzed using most neutral buffers, often with EDTA, a divalent cationchelator that can reduce RNase activity. The use of a conducting fluidthat contains a small amount of 0.3-1% agarose is often helpful formaintaining the alignment of the analyte and cells in the tissuesection. Agarose that is suitable for this use, including lowtemperature melting forms, can be obtained from many commercialsuppliers including FMC 191 Thomaston St., Rockland, Me. 04841 (USA).

Following the addition of the sample and conducting fluid, the twocomponent surfaces of the sensor device are then joined to create a“sandwich” such that their conductive surfaces are brought into contactwith the fluid. In this position each conductive surface of the sensorcontacts the conducting fluid and, in some cases, the sample. Eachsurface is separated from the other by the insulating membrane as shownin FIG. 1. The sandwich is held together by a spring or clamp that isdesigned for this purpose. Care should be taken to prevent theintroduction of bubbles into the sensor as the surfaces are beingpressed together. If present, these can be removed by holding thesandwich sideways and inserting a syringe and needle through the gasketwhile holding the sandwich together loosely over a paper towel or otheradsorbent material. Air and excess buffer will emerge between the platesand flow into the adsorbent. When all air has been removed, the sampleis ready for analysis.

Example 4 Sensors that Can be Heated and Cooled

ITO and other metal coatings have a significant resistance depending ontheir thickness. For most applications the thickness and hence theelectrical resistance of these layers will not be a major concern unlessit impedes the optical clarity of the sensor since relatively littlecurrent flows through the sensor during its operation. The passage oflarger amounts of current through metal coatings can be used to heat thesensor, however, and a preferred means for doing this using glass slidesthat are metal coated on both surfaces is shown in FIG. 7. Slides thatcontain two ITO coatings can be purchased from Delta Technologies. Theyare arranged in the device such that the ITO that is not in contact withthe conducting buffer is used for resistance heating by applying avoltage along the length of the sensor surface. Since this surface doesnot contact the conducting fluid, this applied voltage does not affectoperation of the sensor other than to provide heat. One or both surfacesof the device can be heated in this fashion. The design shown in FIG. 7illustrates a format that can be used to heat both surfaces of thesensor.

Heating the sensor prior to, or during, its operation can facilitateanalysis. Heating prior to analysis can help disrupt the cell membranesin the tissue, thereby enhancing migration of the analytes to the sensorsurface and/or facilitating interactions between the analytes and thedetection reagents. Heating can also contribute to the specificity ofnucleic acid detection. For example, the temperature stabilities ofoligonucleotides as a function of ionic strength are well known. Singlebase changes can result in a substantial change in the stability of anoligonucleotide pair. By heating the sensor surface, the interactionsbetween mRNA and the molecular beacons or PNA can be controlledaccurately. Brief heat treatment can also disrupt the molecular beaconsin a transient fashion, enabling them recognize their “ligands” morerapidly.

Heating can also be used to examine the quality of the sensor surfacebefore use. For example, when sensors that contain molecular beacons areheated above the beacon melting temperature, they will fluoresce. Bymeasuring the amount and uniformity of fluorescence observed, one canmonitor the quality of the coating. Since operation of the beacons isreversible, they will return to their non-fluorescent conformation whenthe sensor is cooled. Heating can also be used to distinguishnon-specific and specific interactions during the analysis of mRNA andother nucleic acid hybridization assays when the sensor is used in thefashion described in Example 2. As the sensor is warmed, non-specificinteractions between mRNA and the fluorescent PNA will be disrupted,preventing the transport of the PNA to the sensor surface. Precisecontrol of sensor temperature can thereby facilitate identification ofsingle base pair mismatches. This may be particularly helpful inidentifying cells that contain mutations in only one allele.

It is also possible to incorporate mechanisms for cooling the sensor.Methods for doing this can be as simple as mounting the sensor on aPeltier heating/cooling stage or as complex as passage of a cooled fluidin a chamber that can be constructed beneath the lower sensor plate orabove the upper sensor plate. By altering the temperature of the sensor,it is envisioned that it can be used for polymerase chain reactionanalyses that can amplify the analytes being studied.

Example 5 Use of an Electrical Field in the Sensor

The sensor has been designed to be operated in the presence of anapplied voltage. While it is conceivable that some analysis can beobtained in the absence of an electrical potential, the benefits ofusing an applied voltage greatly facilitate analysis sensitivity andspeed. Application of an electrical potential to the device canaccelerate the movements of analytes to the sensor surface, depending ontheir charges. This will result in enhanced speed and sensitivity of themeasurements. The presence of an electrical potential can also causedisruption of cells and thereby permit detection of analytes that wouldotherwise be prevented from reaching the sensor surface. Many analysescan be performed under constant voltage conditions. It is not necessaryfor the voltage across the sensor be constant, however, and it willoften be preferable to vary the voltage using patterns shown in FIG. 4a,b or that are found experimentally to be best for a given measurementsystem. The type of polarization pattern to be used is highly sampledependent. That shown in FIG. 4 a is sufficient to enhance interactionsbetween nucleic acids and surface adsorbed molecular beacons. Further byvarying the electrical potential in conditions when the sensor is beingoperated as described in Example 1, it is possible to maintain highconcentrations of analytes near the sensor surface and, at the sametime, prevent them from coming into direct contact with the metal oxide.Since in this location they will be in an ideal position to contacttheir binding partners such as molecular beacons, this can also speedthe reaction.

Variations in the electric field can also facilitate analyses when thesensor is used as described in Example 2. In this case a variation inthe surface charge similar to that in FIG. 4 b is more appropriate. Theuse of a constant electric field has a tendency to promote the migrationof negatively charged nucleic acids to the anode where theconcentrations of fluorescent PNA detection molecules are low. Byvarying the charge on the sensor, the nucleic acids can be made tomigrate through the portion of the sensor that contains the highestconcentrations of PNA. Further if the PNA contain a moderate positivecharge, variation of the potential can cause the paths of the nucleicacids and PNA detection reagents to cross many times. This will enhancethe likelihood that they will interact and speed analysis.

The ability of the sensor to detect protein gene products can also beenhanced by the use of the electric potential. By operating the sensorat the appropriate pH, it is possible to separate protein isoforms thatmay otherwise interact with the same detection molecule. Many proteinscan be phosphorylated, a phenomenon that also results in a shift intheir isoelectric points. Thus, even if two proteins are recognized bythe same fluorophore, they can be distinguished if one migrates towardsthe sensor surface and the other migrates away from the sensor surfaceat the pH at which the sample is being measured. They can also bedistinguished if they are oxidized differently when they come intocontact with the metal oxide coating.

Example 6 Use of the Sensor in a Flow Cell Arrangement as a PerfusionChamber

When the sensor is assembled correctly, the sample will be containedwithin a small chamber the thickness of the gasket. It is possible toattach thin tubes or needles that act as “ports” to access the interiorof the chamber within the gasket. One means of doing this is simply isto insert needles through the gasket. This permits perfusion ofsubstances through the device. Furthermore, it is possible to utilizeboth surfaces of the device for observation. The cell can be used torapidly optimize electrical polarization parameters for promotinginteractions between the analyte and the sensor surface or materialsattached to the sensor surface. Thus, in addition to its use as a sensorper se, it can be used to optimize the parameters needed for analysis oftissue sections in the device to be employed for this purpose such asthat in FIG. 1.

Example 7 Total Internal Reflection (TIR) Illumination of the Sensor

Several methods are available for monitoring analytes in the sensorusing TIR. As noted earlier TIRFM systems can be purchased from Nikonand Olympus Corporations. These enable illumination of the samplethrough either 60× or 100× high numerical aperture objectives that arein optical contact with coverslips that contain the samples. Use ofthese TIRFM systems requires that the surface used for analysis be acoverslip having a thickness of approximately 0.17 mm. They also requirethe use of an immersion oil to make optical contact between theobjective and the coverslip.

Several other types of TIR illumination can be used for examining thesample. A preferred illuminator has the design shown in FIG. 6. Thisdesign permits the sensor to be used in TIRFM with a wider range ofobjectives. Indeed, it is possible to measure fluorescence in thisarrangement using nearly any objective.

The illuminator functions by passing light from a laser through arectangular lens having planar and convex surfaces. This lens is inoptical contact with a triangular prism that is in optical contact witha 0.17 mm coverslip as shown. The prism can also be replaced by a cubeas indicated by the broken lines in FIG. 6. These three components canbe cemented together using Canada balsam or a suitable polymer or theycan be held in optical contact using glycerol. The latter is oftenpreferable since it will facilitate replacement of the coverslip. Themost favorable arrangement of the lens and prism occurs when the focalpoint of the lens is at the junction of the end of the prism and thecoverslip. Since all the light enters the coverslip below the criticalangle, it will be totally reflected within the coverslip until it exitsfrom its edge, which is adjacent to the surface of the sensor surfacethat is to be illuminated. The lens is chosen for its ability to expandthe light from the laser in one dimension. As designed, the illuminatorcannot be moved closer to the side of the sensor. Thus, the lens must bechosen to produce light that is sufficient to illuminate the entirewidth of the sensor.

The illuminator and the sensor are placed upon a microscope stage in aholder designed to keep the illuminator next to the side of the sensor.It is important that the illuminator not be joined permanently to thesensor, however. Microscopic observation across the width of the sensoris accomplished by moving the illuminator and the sensor in tandem asshown in FIG. 6. To observe fluorescence in other portions of thesensor, one moves the sensor along the illuminator, keeping the edge ofthe sensor in contact with the illuminator. By these means it ispossible to scan the entire surface of the sensor. By adding appropriatemotorized drivers, it is contemplated that scanning can be accomplishedin an automated fashion. By keeping a computer record of thefluorescence observed, it should be possible to identify regions ofinterest without the need for immediate observation by the pathologistor surgeon. Retrieval of this positional information from the computercan facilitate human observation and speed diagnosis.

Example 8 Use of the Device with Standard Light Microscopy

The design of the device permits its use with standard light microscopetechniques including phase contrast microscopy, bright field microscopy,darkfield microscopy, differential interference contrast microscopy,confocal microscopy, and epifluorescence microscopy. In most of theseuses, the sample is illuminated by light that passes roughlyperpendicular to the plane of the sensor. This permits examination ofthe entire sample, not just that portion that is adjacent to the sensorsurface. By comparing the images obtained using these techniques withthose obtained by TIRFM, it is possible to identify specific cells thatcontain the analytes being observed during TIRFM even though it is notpossible to observe the entire cell using TIRFM.

The tissue sections can also be stained to increase the contrast betweenvarious cell types or organelles. This can be done using non-fluorescentdyes prior to TIRFM. It is also possible to use fluorescent dyes priorto TIRFM if the dye recognizes a substance to be analyzed or if the dyecan be excluded from the evanescent field by application of the electricfield. The advantage of using a dye before performing TIRFM is that itwill facilitate correlating specific cell types with the location of thefluorescence. In some cases, however, it may not be possible to stainthe tissue prior to TIRFM. In this case, it may be necessary to removethe non-sensor surface from the device to gain access to the tissuesection. This can be facilitated by including a small layer of gauzebetween the non-sensor surface and the tissue section to preventsticking of the surface to the tissue.

In some cases it will also be useful to employ the electrical potentialthat can be generated by placing a charge on the sensor surface toremove excess stain from the tissue section, thereby reducing the timeneeded for staining and clearing the background. This can be done byplacing the sensor surface and its attached tissue section in a bath andapplying a low voltage across the sensor surface and the bath.

Example 9 Use of Photobleaching within the Device

One of the limitations of using fluorescence to study gene expression isrelated to the number of fluorophores that can be distinguished at onetime. Photobleaching can expand the measurement range, however. Forexample fluorescein and Alexa Fluor488 have about the same fluorescencespectra. The former is much more readily photobleached, making itpossible to distinguish analytes that are labeled with fluorescein fromthose labeled with Alexa Fluor488 by the differences in the rates atwhich they are photobleached. The combined use of organic dyes andquantum nanodots, which are nearly impossible to photobleach shouldextend this technique further.

Example 10 Use of the Device to Measure Enzymes

Another use of the device is for measurements of enzyme levels in tissuesamples. Many cancers have different levels of extracellular andintracellular proteases and these can be readily distinguished by use offluorophores that contain protease cleavage sites. Cleavages at thesesites by the actions of the specific proteases will cause the release ofa quencher from the fluorophore resulting in fluorescent light emission.One of the advantages of the device described here is that it ispossible to use the electrical potential to cause proteins and othermolecules that are not nearly as negatively charged as mRNA and nucleicacids to migrate to a different sensor surface than the nucleic acids.This will permit simultaneous analysis of mRNA and proteins in the samesample. Application of similar approaches will permit the measurement ofany type of enzyme reaction that can lead to the appearance ordisappearance of fluorescence.

The ability of the sensor to detect differences in the net charge of amolecule can also be used in assays of kinases and phosphatases, enzymesthat alter the phosphorylation status and charge of a molecule. Forexample it is possible to prepare fluorescent peptides that aresubstrates for various protein kinases. The presence of kinase activityin the sample can cause the fluorescent peptide analog to migrate to theanode whereas the non-phosphorylated analog may fail to migrate or maymigrate to the cathode at the pH employed in the conducting fluidbuffer. This will permit cell specific analysis of these importantcellular enzymes, many of which have been implicated in tumorigenesis.

The ability of the sensor to detect differences in charge can also beused to detect protease activity. Fluorescent protease substrate canreadily be designed such that proteolysis will change the ability of thefluorophore to migrate to either the anode or the cathode, where it isreadily detected. This can be accomplished by adding charged amino acidresidues to the substrate, which are then cleaved by the protease.

Example 11 Use of the Device to Measure Small Molecules

Binding of small fluorophores to proteins or larger macromoleculesresults in a loss of molecular mobility. When the small molecules arelabeled with fluorophores, this will result in a change in fluorescencepolarization that is readily detected. The device illustrated in FIG. 1can also be used to monitor changes in fluorescence polarization andthereby be used to monitor the levels of small analytes in tissuesections. In this case, it is often desirable to coat the sensor surfacewith antibodies that are specifically capable of recognizing theanalyte. One means of attaching the antibodies to the surface involvesbiotinylating them and then coupling them to the surface through astreptavidin bridge. Methods for biotinylation of antibodies and otherproteins are well-known in the art.

Example 12 Use of Multiple Molecular Beacons to for Cell Classification

As noted earlier, data obtained using microarrays suggest that many mRNAwill be elevated at the same time in cancerous and malignant cells. Thisphenomenon can contribute to the sensitivity of the device. Molecularbeacons that are specific to multiple mRNA are coupled to the surface ofthe sensor surface as in Example 1. When these are labeled with the samefluorophore, they will detect the increase in any of these mRNA.Similarly, some populations of mRNA decrease in cancerous cells. Bymixing these and labeling them with a different fluorophore than used inbeacons to monitor mRNA whose expression is found to be unchanged andwith a different fluorophore that used in beacons designed to monitormRNA whose expression is found to be increased, it is possible toincrease the sensitivity of the method. As noted earlier, it is alsopossible to make use of both surfaces of the device to increase thenumbers of analytes that can be monitored. Similar types of mixtures canbe employed for analysis of gene transcription produces using the sensoras described in Example 2.

Example 13 Use of the Device for Electrophoretic Separation of Samplesin Three Dimensional Electrophoresis

The principles shown in the device illustrated in FIG. 1 can also beapplied to techniques other than analysis of tissue sections. One use ofthe device is to separate small quantities of materials byelectrophoresis. For example, when the ends of the device are left open,it is possible to pass an electrical current from one end of the deviceto the other by attaching electrodes to each end. If the device isloaded with polyacrylamide gel or other medium used to separateproteins, nucleic acids, or other substances by electrophoresis, samplesthat are placed in the gel will separate according to their netcharge/mass ratio. Thus, it will be possible to separate proteins bytheir isoelectric points in a gel that contains a pH gradient. It willbe possible to separate proteins by their molecular weights in a gelthat contains sodium dodecylsulfate (SDS). It will also be possible tooperate the sensor in a two dimensional fashion by alternately passingcurrent through the ends of the device and through the sides of thedevice. This will permit two dimensional analysis of trace quantities ofanalytes. Following separation, the separated analytes can be forced tomigrate to one or both surfaces by passing an electrical current betweentheir component metal oxide layers. When the proteins or analytes reachthe surface they can be detected using fluorescence assays performedusing the apparatus in a TIRF or TIRFM mode. One use for this procedurewill be to analyze extremely small samples, such as the components of asingle cell or nucleus. Once the locations of the analytes on thesurface are identified by their fluorescence of their influence on thefluorescence of materials attached to the surface, they can be removedand identified further by mass spectroscopic or other methods.

Example 14 Use of the Sensors in a Microtiter Well Plates

Microtiter plates are often used for analysis and the application of anelectrical potential to this assay format can facilitate analysis. Forexample, it can be used to increase concentration of an analyte at theplate surface. It can also be used to reduce the concentration of ananalyte at the plate surface. Many of the applications of the sensorsexcept for those that involve tissue sections can be transferred to amicrotiter well plate format. These include enzyme assays and nucleicacid assays. Several formats can be used to build microtiter plates thatcan be used with electrical potentials. One of these formats isillustrated in FIG. 8.

Example 15 Sensors with Permeable Optical Polymers Polymeric Hydrogels

One of the limitations of the sensor shown in FIG. 1 is related to thelocation of the electrodes, which limitation consists of the ITO coatingon the glass surfaces. These coatings are situated between the surfaceof the specimen being examined and the optical surface. While the metalinterferes only slightly with the optical quality of the surface, thefact that it contacts the fluid between the sample and the area wherethe sample is being examined limits the amount of voltage that can beapplied. This voltage should be kept below that which will causeelectrolysis of water, a phenomenon that will produce bubbles andinterfere with migration of the analytes thereby hindering analysis.Furthermore, an excessive potential can have a negative impact on theanalyte if the analyte contacts the metal electrode surface, which isalmost certain to occur. This limitation on the amount of voltage thatcan be applied in the device can impede the analysis by preventingefficient and uniform extraction of material from the cells. It would bepreferred to locate the metal electrodes on the opposite surface of theglass from that shown in FIG. 1 where electrolysis of water would notinterfere with analysis and the sample would not come into contact withthe charged metal coating. Unfortunately, however, doing so caninterfere with the uniformity of the electric field near the opticalsurface, a phenomenon that can cause uneven migration of the analyte. Asa consequence of placing the electrode on the opposite surface of glassfrom that shown in FIG. 1, the uneven deposition of analyte on theoptical surface may interfere with the correlation of the distributionof the analyte on the glass surface with that in the tissue section.

The voltage limitation of the sensor can be overcome by replacing theglass optical components of the sensor with permeable optical polymers(polymeric hydrogels) that are permeable to ions and placing the polymerbetween the sample and the electrodes as shown schematically in FIG. 9A.Consequently, the electrolysis of water will not interfere with theanalysis and the analytes will not come into contact with theelectrodes. This will permit the use of greatly increased voltages forelectroporation of analytes from the tissue section and migration ofanalytes to a region where they can be analyzed. Permeable opticalcomponents (i.e., those that refract light) can be made of a variety ofpolymers. The properties of these polymers are well known and haveenabled the construction of contact lenses that can be worn for extendedperiods. Furthermore, these polymers can be designed to have manychemical features that will facilitate their use in the sensor. Forexample, they can be composed of materials that have either a netpositive or net negative charge or that have the capacity to buffer thepH of the area in which they are located. This can be used to alter thecharge of the analyte and change its mobility within the device. Thesepolymers can also be designed to have a refractive index that willenable the use of total internal reflection, a property that rendersthem useful for the analysis of trace amounts of materials includinganalytes from tissue sections.

The use of polymeric materials has another major advantage as well. Itpermits the design of components that include aqueous solutions that canbe stored in sealed pouches. This frees the operator from having to addwater or buffers. This is important because it lessens the potential formistakes to be made. When tissue sections are being made during surgery,time is of the essence. The fewer operations that are required, the lesslikelihood that mistakes will be made. Furthermore since, the fluidcomponents are within the gels, it reduces the chances that bubbles willbe introduced between the tissue sections and the components of thesensor when assembling sensor components, a process likely to be donemanually by the person cutting the tissue sections.

The overall principles that underlie the operation of a polymer-basedsensor are the same as those that are responsible for the operation ofthe sensor in FIG. 1. In both devices, the analysis depends on the useof an electric field to cause analytes to mix with a detection reagentto form a complex that can be detected optically. The design of thepolymer-based sensor illustrated in FIG. 9 differs from that in FIG. 1in the location of the electrodes relative to the optical surface thatis being used for detection. In FIG. 1, the electrodes are between thesample and the surface. In FIG. 1 the optical surface is between thesample and the electrodes. Another difference in the sensor illustratedin FIG. 9 and that in FIG. 1 is that the optical surface in FIG. 9permits current to flow through it; that in FIG. 1 blocks the flow ofcurrent.

The use of polymers in the design of the sensor in FIG. 9 permits it tobe constructed in a modular fashion. As shown in FIG. 9B, the sensor canbe arranged into two parts, which will be termed the anode and cathodesensor assemblies to reflect the assembly that will contact with theanode and cathode respectively. These can be marked with a color code,e.g., red for anode and black for cathode, to make them more easilydistinguished. This is particularly useful when they contain polymersthat differ in composition and/or buffer content. There is no particularorder in which the sensor needs to be assembled in most cases or for theanode assembly to be on the bottom and for the cathode assembly to be onthe top. Thus, the sample can often be applied to the anode sensorassembly before addition of the cathode sensor assembly. It is usuallybest to do all the operations in the same fashion, however, to avoidmaking mistakes such as using two anode assemblies or two cathodeassemblies when the compositions of these are not identical. The chancesfor making this mistake are reduced by the design of the apparatus thatis to be used for electrophoresis, which is incapable of being loadedwith two anode or two cathode assemblies. Furthermore, the design of theanode and cathode assemblies (FIG. 9D) and this box (FIG. 9C) makes itimpossible for the anode and cathode assemblies to be reversed.

The anode sensor assembly contains the polymer that will be the primarysite of analysis when RNA gene products are to be examined from tissuesections since this is the direction in which these gene products willmigrate during electrophoresis. This is identified as component #3 inFIG. 9A. The polymer that is located adjacent to this, that is shown ascomponent #4 in FIG. 9A, is where most of the combination of the RNA andthe detection reagent will occur. The polymer in component #4 is usuallyof a lower refractive index than that used to construct component #3since this will permit illumination of the polymer in component #3 bytotal internal reflection. As a result fluorescent material that remainsin component #4 will not be illuminated and, therefore, not interferewith the analysis. Since the ability of the polymers in component #3 and#4 to buffer the pH can be made to differ, this property can be used toalter the net charge on the fluorescent detection reagent and therebyprevent it from entering the polymer in component #3 unless it is boundto negatively charged RNA. For example, if the detection reagent has anet positive charge at the pH of the buffer in component #4, it willmigrate towards the cathode and cross the path of the RNA that ismigrating from the tissue section towards the anode. This will increasethe sensitivity of the method by minimizing the background due to thepresence of unbound detection reagents. It will also permit the use oflarger concentrations of detection reagents, which will increase thechance that they will interact with species of RNA that are beingmeasured.

When the sensor is being used to measure RNA and no other gene products,virtually all the measurements will be made on the part of the sensorshown as component #3 in the anode assembly (FIG. 9). This simplifiesthe design of the cathode assembly, which can consist of a singlepolymer, a spacer (component #8) and the electrode (component #9). Theprimary function of the cathode assembly in this case will be to delivervoltage across the device. It should be noted, however that the cathodeassembly can also be used to make measurements of analytes that arepositively charged. In this case one will want to include a polymer thatcan be used for optical analysis as outlined in FIG. 9. Furthermore, itis possible to use the cathode assembly to facilitate staining of thetissue sections with positively charged dyes. The polymer to be used inthe cathode assembly should be of optical quality even when it is notbeing used for analysis, however. This is to permit visualization of thetissue section after electrophoresis and analysis of the RNA iscomplete, a requirement that will become clear later.

It should also be noted that sensors can be made with molecular weightcut off devices by inserting a piece of dialysis tubing between thepolymers. When these are placed between components #2 and #3, all thehigh molecular fluorescent species will be collected at a surface thatcan be made very thin to permit better detection (c.f., component #2 a,FIG. 9A). The dialysis tubing is useful for RNA analysis since itprevents it from passing through component #3 and being lost.

The sensor also contains other components that are not optical polymersor even polymers but that are present to facilitate delivering anelectrical potential to the sensor. Components #1 and #9, which serve asthe anode and cathode, respectively, are designed to create anelectrical potential across the device. Components #2 and #8 can beincorporated into the anode and cathode as shown in FIG. 9D. The anodeand cathode elements shown in FIG. 9D are constructed from a thin stripof conducting metal, which serves as the back and a molded piece ofclear plastic, which serves as the bottom and sides. A piece of sinteredpolyethylene is used to make the front and after the device is loadedwith fluid, to create the top cap. The sintered polyethylene frit at thefront provides the support needed for the polymer gel (i.e., eithercomponent #3 or #7 shown in FIG. 9A). The top of the device issurrounded by a piece of heat shrink plastic to seal this portion of thedevice. This is removed during use although in some cases the amount ofbubbles is not sufficient to increase the pressure in the electrodecomponents to a point in which it interferes with analysis. In thiscase, it is not essential that the heat shrink plastic be removed.

The final steps in the construction of the anode and cathode assembliesinvolve layering the polymers illustrated as components #3 and #4 andcomponents #6 and #7 on the anode and cathode respectively. This isshown in FIG. 9E. Note that when RNA species are being monitored, it isuseful to insert a piece of dialysis tubing between the anode andcomponent #3. This can also be accomplished by polymerizing component #3on top of a piece of dialysis membrane having a pore size sufficient toblock the migration of RNA. This will trap any RNA-fluorescent complexesthat are migrating through component #3. Further, since this can bequite thin, it can increase the resolution of the device. The presenceof the dialysis tubing is not essential, however. Several other means oftrapping the complex are also possible and these can be attached to thepolymer. Once the gels have been added to the device as seen in FIG. 9E,then the device is enclosed in an airtight bag. A few drops of water or,preferably, a water-saturated piece of towel is added to make certainthe device remains moist until use. All steps in the preparation of theassembly should be done under clean conditions to prevent bacterial orother contamination. Also, since the device will be used to measure RNA,care should be taken not to contaminate the device with RNase. Thismeans that persons assembling these components should be wearing glovesand taking standard precautions for working with RNA containingmaterials. The device can also be sterilized by ethylene oxide beforethe bag is sealed to prolong the half-life of the assembly.

Use of the sensor device requires only a few simple steps. Either theanode or cathode assembly pack is opened at the time of sectioning or asection is placed directly on the exposed gel. When this is opened justprior to use, there should be sufficient moisture to give good contactof the tissue section to the gel. It is important that no air be trappedbetween these sections, however, since this can interfere with RNA orother analyte extraction from the tissue. A few drops of sterile watercan be added at this time to avoid this problem, if needed. Once thesection has been placed on the anode or cathode assembly it is coveredby a cathode or anode assembly, which is placed on top of the sectionsuch that its gel contacts the section. It is a good practice to beginwith either the anode assembly since it is easy to see how the tissuesection contacts the polymer and since this contact is the mostimportant. Then, one adds the cathode assembly such that its gel sidefaces the tissue section. Again, a few drops of water might be needed,but this should not be necessary if the assembly package is opened atthe time of use and if it has stayed hydrated.

Once the sensor sandwich has been assembled, it is ready for theelectrophoresis step. The sensor sandwich is inserted into theelectrophoresis chamber as diagrammed in FIG. 9C. The cutouts on thesandwich prevent the electrode from being inserted into theelectrophoresis box in an improper orientation. They also guard againstmistakes in assembly such as the preparation of the sandwich from twoanode assemblies or two cathode assemblies. The electrophoresis isperformed at voltages of up to 100 volts/cm. The actual voltage usedwill depend on the tissue with soft tissues requiring lower voltagesthan tissues that contain substantial amounts of connective tissue. Itis often useful to use a transient voltage that is very high to causeelectroporation of the cells, which will release the RNA. The limit tothe amount of voltage that can be employed depends on how the tissue isto be examined after the gene products have been detected. The use ofvery high voltages tends to destroy the tissue, making it more difficultto study after electroporation. This can be reduced by the inclusion ofsmall amounts of detergent in the polymer layers that are in contactwith the tissue section.

Following electroporation and electrophoresis, the sample is ready forvisualization. This is done by removing the sandwich from theelectrophoresis box and, in the case of negatively charged analytes suchas RNA, observing the fluorescent material that is collected in theportion of the sensor in components #2 a or #3 (FIG. 9A). As seen inFIG. 9F, anode components #1 and #2 are removed from the sandwich. Thesandwich is then placed on top of a fiber optic window or a fiber optictaper that is covered with a piece of Dupont FEP film or other film oflow refractive index. It can also be covered with water when a dialysismembrane is included, but this can risk contamination of the window ortaper. This can be avoided by covering it carefully with some microscopeimmersion oil and a thin coverslip, which can be replaced if it getscontaminated. The presence of a low refractive index material betweenthe sensor and the window or taper is required to minimize unwantedstray light entering the detector from the illumination source. Mountinga cutoff filter beneath the FEP film can also reduce the stray light,but this will also lower the sensitivity of the device. The other end ofthe fiber optic window or fiber optic taper is mounded on the sensorchip of a charged coupled device (CCD). Mounting is performed in such away that the dialysis tubing (when present) or component #3 is incontact with the Dupont FEP film directly on top of the fiber optic. Thesample is illuminated through the side of component #3 using lasers orother light sources that have the appropriate wavelength. Due to thefact that the refractive index of component #3 is greater than that ofthe adjacent polymeric gel or the Dupont FEP film, the excitationwavelengths will be reflected within the gel internally where it iscapable of illuminating fluorescent material that has become associatedwith the analyte. Consequently, none of the unreacted fluorophoredetection reagent that remains in other portions of the apparatus willbe illuminated and all will remain invisible.

Example 16 Sensors with Peptide Nucleic Acids PNA

A desirable detection reagent for a nucleic acid is a molecule that hasbases that are held in an ordered fashion such that they can formWatson-Crick base pairs with nucleic acids and that lacks the negativecharges in the backbone atoms that hold the bases in order. This isbecause the negatively charged phosphates of nucleic acids exert arepulsive effect on formation of the oligonucleotide duplex. Byreplacing the negatively charged phosphate atoms with atoms or groups ofatoms that have either no charge or that have positive charges, one candevise detection reagents that will have high affinity for specificoligonucleotide sequences. Indeed, the affinities of these can begreater than that of nucleic acids for complementary nucleic acids.

PNA are molecules capable of forming Watson-Crick base pairs withnucleic acids that have a peptide backbone. Because they lack thenegatively charged sugar-phosphate backbones found in RNA and DNA,hybrids of RNA-PNA and DNA-PNA are known to be highly stable [24]. PNAcan be constructed to be essentially uncharged, negatively charged, orpositively charged simply by incorporating amino acids into theirbackbones by standard peptide synthesis chemistry. PNA have also beenlabeled with fluorophores [18,21] and used to detect nucleic acids byfluorescence in situ hybridization (FISH). PNA are not the onlystructures that can be used for this purpose, however. Agents in whichthe phosphate is replaced by sulfur or carbon are also useful.

When PNA are bound to nucleic acids, they can alter its mobility [20] ingels or in capillary electrophoresis tubes [17]. The ability of DNA tochange the charge of PNA such that its migration in an electric field isreversed has not been employed, however. This is a particularlyimportant property for use in a sensor of the type taught here in whichit is preferable for the electrophoretic migration distance to berelatively short. Binding of uncharged or positively charged PNA to RNAor DNA will cause it to become negatively charged. As a consequence, thecomplex will migrate in the opposite direction from the uncomplexed PNAin an electric field. This can be used to separate bound PNA fromnon-bound PNA. If the PNA is labeled with a reagent such as afluorophore, a radioisotope, biotin, or other molecule that does notcause it to acquire a net negative charge, then binding of the labeledPNA to nucleic acids will cause it to be separated from the non-boundPNA. This provides a very useful and simple tool for the identificationof nucleic acids. Further, this permits the labeled PNA to be employedat very high concentrations, which facilitate its interactions withnucleic acids without increasing the background signal when the signalis measured by a technique such as total internal reflectionfluorescence of TIRFM. In addition, this property can be used to causenucleic acids or other charged materials to migrate into areas wherethey can be assembled into complexes.

Because PNA have a peptide backbone and can be synthesized similar topeptides, it is possible to incorporate several different types oflabels into them. For example, it is possible to add cysteine residuesto PNA that will permit labeling of the molecule with fluorescent probesthat react with thiols or that can be made to react with thiols. Manysuch probes are available from Molecular Probes, Eugene, Oreg. It isalso possible to incorporate lysine molecules into PNA. This will givethem a positive charge or serve as a labeling site for amino reactiveagents. These are also available from Molecular Probes in a wide varietyof absorption and emission wavelengths. One can incorporate arginineresidues into PNA to alter their charges as well. PNA have also beenlabeled with histidine residues [24]. The pK of the imidazole moiety ofhistidine can have a favorable influence on the migration of PNA in anelectric field that has a pH gradient. For example, at low pH, histidineis positively charged. At high pH, it becomes uncharged. A PNA thatcontains histidines will tend to migrate away from an anode when it isin a low pH environment. Its mobility will be reduced as it reaches ahigher pH environment due to the loss in charge. Thus, one can easilydevise conditions in which histidine labeled PNA migrate away from ananode until they reach a region of an electrophoresis chamber in whichtheir migration becomes slow. One use of this is to drive the PNA to aregion of the chamber away from the anode but prevent them frommigrating to a region where they would be unable to react witholigonucleotides. PNA that have bound to oligonucleotides will migrateback towards the anode away from their non-bound counterparts.

The design of PNA is relatively straightforward and is based on thenotion of Watson-Crick base pairing [24]. The fact that PNA areuncharged or can be made positively charged enables them to invade shortRNA-RNA duplexes found in most gene expression products. Increasing thetemperature of the device can facilitate this. The usual length for thehybridization reaction is 16-25 bp. The only other considerations indesigning the PNA relate to the solubility of the molecule. Longuncharged PNA are generally not soluble and are not well suited for usein the sensor. Positively charged PNA are much more soluble and muchbetter suited for the measurements with the sensor, particularly iftheir charges can be modulated as a function of pH, e.g., by addition ofresidues such as histidine when they are employed at pH values in therange of 6-8.

A key to the operation of the sensor is its ability to maintain a verylow background. This enables the detection of trace quantities of RNAanalytes. As just discussed, the use of PNA and the ability to reversethe migration of labeled PNA molecules in an electric field is one meansof maintaining a low background. Another method of reducing thebackground is to use PNA that have a hairpin conformation similar tothat found in molecular beacons. In the PNA hairpin conformation [23],which is found before the PNA is complexed with an oligonucleotide, thefluorophore at one end of the PNA is quenched by a molecule that isattached to the other end of the PNA by resonance energy transfer. Thisoccurs due to the proximity of the fluorophore and the quencher, whichare near one another only when the PNA has a hairpin conformation.Binding of the PNA to RNA causes the hairpin to become linear, whichresults in the fluorophore being moved from the quenching agent. As aresult, the fluorescence becomes visible and can be observed. Sinceformation of the hairpin shape does not alter the isoelectric point ofthe PNA before it is bound to RNA, the hairpin shaped PNA will alsomigrate away from the anode. This will change when it interacts withRNA, however, the time that the fluorescent PNA-RNA complex will bemigrating towards the anode. These movements are illustratedschematically and described in FIG. 10.

Another important means of reducing the background fluorescence is theuse of total internal reflection optics. By restricting the illuminationto the components of the sensor that contain the fluorescent RNA-PNA*complexes, it is possible to prevent illuminating the uncomplexed PNA*,which would contribute to the background. It is desirable to illuminateonly component #3 in the sensor. This can be done if component #3 istransparent to the illuminating radiation, if component #3 has a higherrefractive index than component #4, and if component #3 is illuminatedat an angle less than the critical angle. This can be calculated fromSnell's law from the refractive indices of components #3 and #4.

The requirement for total internal reflection illumination of component#3 can be met using polymers that have been designed for theconstruction of soft contact lenses that are intended for long use.These have been designed to be sufficiently porous to enable air andfluids to pass through the lens where it can reach the cornea. Further,their refractive index is sufficient to bend light needed for visioncorrection. The refractive index of these materials has been shown topermit their use for total internal reflection fluorescence [22] aswould be expected from their refractive indices.

There are several materials that have been used to construct contactlenses. Two of the most common are HEMA (hydroxyethylmethacrylate) andHEMA-MAA (HEMA-methacrylic acid). Commercially available lenses of theformer have a refractive index of approximately 1.437 and contain 42%water. Commercially available lenses of the latter have a refractiveindex of approximately 1.407 and contain 55% water. The latter are alsomuch more permeable and have significantly larger pore sizes. Thus,these materials are suited for both total internal reflection in aqueousbuffers in which the refractive index is approximately 1.33-1.37 and forelectrical conduction needed for electrophoresis. Both types of polymerscan be readily molded and made sufficiently thin for use in the deviceand are commonly made in sizes in the range of 0.2 mm. Due to thedesirability of having the most resolution possible, it is importantthat the thickness of component #3 be kept relatively small, in theorder of 0.2 mm. The thickness of component #4 should also be keptsmall, but this is not as important as that of component #3, which isthe component that will be illuminated. Since component #4 is not to beilluminated, its composition is much less critical than that ofcomponent #3. In fact the composition of component #4 can be virtuallyany soft gel that can be molded into a shape that will fit betweencomponent #3 and the tissue section. The critical features of component#4 are that it permit migration of RNA, PNA*, and RNA-PNA* complexes andthat it have a lower refractive index than component #3 to permitcomponent #3 to be illuminated by total internal reflectionfluorescence. Thus, it is even possible to use a low percentagepolyacrylamide or agarose gel for component #4. The use ofpolyacrylamide also permits the incorporation of immobilines into thegel during polymerization [25,19]. These should be chosen to buffer thelocal pH such that PNA* will be positively charged and will migratetowards the tissue section and away from component #3. The immobiline tobe chosen, if one is to be used, would depend on the design of the PNA*,which will depend on the RNA to be monitored. In general, it is mostuseful to chose an immobiline that will buffer the pH of component #4 tobe at least 0.1 pH unit less than the pI of the PNA*. The pH of thesolution that is in components #1-3 should also be lower than the pH ofthe immobiline in component #4.

When RNA is the only cellular constituent to be analyzed, thecomposition of components in the cathode assembly is not nearly ascritical as those of components #3 and #4. In general, components #6 andabove should be at a pH that is equal to or greater than that ofcomponent #4. These can be fabricated of polyacrylamide or HEMA-MAA.When component #7 is to be used for total internal reflection, it isbetter to construct it of HEMA. It will be subjected to the sameconsiderations as those discussed next for component #3. Note, that whenan immobile is not used, the buffer throughout the sensor should have apH that lower than that of the pI of the PNA*.

The design of component #3 should be considered carefully since this isthe component of the sensor that will be illuminated and used fordetecting the sample. As a rule, component #3 should be a hydrogelhaving an optical density greater than that of the buffer on either sideof it and greater than that of component #4 to permit its illuminationin a total internal reflection fashion and since it should be capable oftransmitting an electrical current. This is a property that is alsofound in most soft contact lens hydrogels such as those that containHEMA. Methods for preparing polymeric hydrogels containing HEMA andother substances are well known in the art and more than 700 patentsrelated to the fabrication of these types of polymers were obtained bysearching the United States patent data base with the terms “HEMA” and“contact lens.” Particularly useful United States patents are numbers6,447,118, 6,552,103, 6,582,631, and 6,623,747, which describe methodsfor molding and modifying hydrogels that can be used to preparecomponent #3 in the sensor using an appropriate mold. It should beappreciated that nearly any hydrogel material that is has a refractiveindex that is sufficiently greater than the buffer to be used to permittotal internal reflection of light, that has the ability to conduct anelectrical current, and that is optically clear at the wavelengths oflight used for illumination and fluorescence will be appropriate for usein sensor component #3 and for use in sensor component #7 when thelatter will also be used for analysis and illuminated by total internalreflection.

Several aspects of component #3 can influence on the operation of thesensor. For example, when PNA having positive charges are used to detectRNA, it is useful to make the surface of component #3 positivelycharged. This will facilitate migration of non-complexed fluorescent PNAaway from the surface of component #3 into areas of component #4 thatwill not be illuminated by total internal reflection fluorescence. Whenthe sensor is used to detect RNA, it is also useful to fabricatecomponent #3 from a hydrogel that has a smaller pore size. This willenable component #3 to behave in a semi-permeable fashion and therebyprevent the RNA-PNA* complex from migrating through it. This will avoidthe need to attach materials to component #3 that are capable of bindingnucleic acids or to use a semi-permeable membrane such as component #2 a(FIG. 9A). Since hydrogels that have smaller pore sizes and that containless water have an increased refractive index, this can facilitate thedesign of total internal reflection optics that will be used duringanalysis. Another aspect of the design of component #3 relates to itssurface that faces component #2. When the detection system will involvethe use of a fiber optic window or a fiber optic taper, component #3 maycome into contact with the fiber optic. Since the fiber optic will alsohave a high refractive index, this could create the potential for thelight being used for illumination to pass directly into the fiberthereby causing a high background and possibly preventing detection oflight from the RNA-PNA* complex. Thus, it is essential to have a thinlayer material of lower refractive index between component #3 and thefiber optic. This can be provided by placing the Dupont FEP film betweenthe fiber and component #3. It can also be provided by a thin layer ofbuffer that can be attached to the surface of component #3 that will benearest the fiber optic. For example, if the fiber optic is coated witha hydrophobic silicone monolayer such as Sigmacote purchased from SigmaChemicals (St. Louis, Mo.), the surface of component #3 facing the fiberoptic can be designed with an oligosaccharide coat to retain a smalllayer of water that will separate it from the fiber optic. This issufficient to cause total internal reflection from this surface. Theseproperties of component #3 are indicated in FIG. 11.

Following completion of the electrophoresis, it is necessary to detectthe fluorophores that are bound to the surface of component #3 or, ifthe pores of component #3 are sufficiently large, that have traversedcomponent #3 and accumulated on component #2 a. This can be accomplishedusing an illuminator that is focused on the side of component #3 as seenin FIG. 9. Lasers are the most useful types of illumination for thispurpose since they can be used as a source of coherent monochromaticlight that can be focused to a small size. When more than one color offluorophore is to be examined, the type of illumination that is to beemployed will depend on the manner in which the signal from the sampleis to be detected. When this is a camera based detector that employsthat has a fiber optic window or a fiber optic taper, it is useful toemploy an illuminator that is capable of illuminating component #3 atmultiple wavelengths. This is because it is important to keep thedistance between the fiber optic and component #3 as small as possible,making it desirable not to insert different filters between the sampleand the fiber optic. To detect multiple colored fluorophores, one wouldemploy multiple lasers or a dye laser that can be used with differentdyes to produce desired wavelengths. By illuminating with the longerred-most wavelengths followed sequentially with wavelengths that areincreasingly shorter, it is possible to obtain multiple pictures of thesample and to resolve these into different colors. A diagram showingthis is illustrated in FIG. 12. A camera-based detector that employs anobjective to monitor the fluorescent samples that are illuminated incomponent #3 is readily adapted for use with filters. Thus, one can varyboth the excitation wavelength and the emission wavelength. Theadvantage of the fiber optic based system is that it recovers much moreof the fluorescent light and can be designed to detect fluorescence fromthe entire sample at one time. This increases the sensitivity ofdetection and speeds the analysis substantially. This is usuallysufficient to offset the greater flexibility gained from the use ofemission filters that are more easily introduced into the objectivebased design.

Once the fluorescent image of the gene products has been captured,components #8 and #9 can be removed (if they have not already beenremoved) and the remainder of the sensor sandwich can be transferred toa light microscope. This permits visual inspection of the tissuesection, if desired. Alignment of the section with the fluorescenceimage can be made by comparing the position of component #3 while thesection is on the microscope stage with that while it was on the fiberoptic. This is because the position of the tissue section will remainconstant with regard to the position of component #3.

The sintered materials that can be used to make components #2 and #8 canbe obtained from SPC Technologies Ltd., 1 Raven's Yard, NethergateStreet, Harpley, Norfolk, PE31 6TN, UK.

DETAILED DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate an overview of the sensor device showing thesensor from three different perspectives. FIG. 1A shows an end view ofthe sensor device 100. Sensor device 100 comprises tissue sections orother analytes 101, brass or other conductor 102, conducting tape 103,ITO or SnO2 coated slides 104, gasket insulator 105, matrix (includingbuffers) 106, microscope objective 107, CCD camera or other detector108, and voltage generator 109.

Since a primary use for the sensor will be microscopy, the example shownhere is constructed from microscope slides. There is no reason thatlarger or smaller sensors cannot be made, however. The sensor can alsobe constructed of 1 mm thick slides, a common size for microscopy,coverslips that are 0.17 mm thick, a common size for microscopy, or froma combination of the two. Indeed, since the device is likely to beobserved by TIRFM, a preferred construction would involve the use of acoverslip for the portion of the sensor most likely to be viewed usingTIRF. When RNA gene expression products are to be examined, this will bethe anode. The view in FIG. 1A is of the sensor from the end. The slides(shown in solid gray) are coated with ITO, SnO₂, or other conductivemetal. The thickness of this layer is not critical as long as it isthick enough to conduct current and thin enough to permit tissues to beviewed. The location of the coating is illustrated as a red line. It canbe difficult to attach electrical leads to the metal coating of theslide. To make the sensor more robust during handling needed to load itwith tissue sections, it has been designed to fit into metal holder thatis made of brass or other conductor (gray oblique lines that riseupwards). The thickness of this holder is not important to the functionof the sensor but should be sufficient to withstand rough handling in anoperating room setting. The leads that control the potential on thedevice (black lines) are soldered or otherwise attached securely to thebrass conductor. Contact between the metal coating and the conductor ismade via a brass tape that is wrapped around the electrode (blackrectangular shape). This is held to the slide by a glue that is stablein the autoclave, enabling the device to be sterilized. The two portionsof the sensor are separated by a gasket (green), which serves as aninsulator. The composition of this gasket is not critical but it is bestif it is of a rubbery consistency, which makes it easier to use and tokeep the device from leaking. Gluing the gasket to one sensor makes thedevice easier to load. Several other designs are possible so long asthey result in a device that is able to deliver an electrical potentialacross the tissue section (shown in speckled contrast). Observation ofthe material can be from the bottom as shown here or from the top.

FIG. 1B shows a top view of the sensor device 100. Sensor device 100comprises brass or other conductor 102 (Note that this has a shape thatpermits it to contact the conducting tape with which it forms anelectrical contact and at least one of the conductors has a hole thatpermits observation of the metal coated slides and the material that issandwiched between them), ITO or SnO2 coated slides 104 (Note that thetape is folded around the edge of the slide such that it makes contactwith both surfaces), and gasket insulator 105 (Note that this shapepermits it to contact the conducting tape on the sides of the deviceand, in cases in which a fluid is present, the slides at the end of thedevice).

The sensor contains at least one and preferably two opticallytransparent components. These are covered with a tape that is foldedaround the sensor as indicated in the first image of the top view. Othermethods of attaching the electrical contacts will also work, but thisdesign was chosen for its robustness, high conductivity, and ease ofconstruction. Note that the conducting tape lies along the top andbottom of the entire sensor surface to facilitate even electricalcontact with the metal oxide layer and the brass conductor. The edge isnot coated throughout most of the slide, however, leaving it availablefor TIRF illumination. There are other means of attaching the tape suchas running it along the metal oxide layer and folding it back around theends. The method of attaching the tape does not matter to the functionof the sensor, provided that the edge of the plate will permit TIRFillumination, should this type of illumination be used during analysis.Shown below the slide is the structure of the conductor and the gasket.Basically, each has a rectangular shape that enables it to contact theconducting tape without blocking the ability of the user to observe thecontents of the sensor, e.g., tissue sections.

FIG. 1C shows a side view of the sensor. FIG. 1C shows an end view ofthe sensor device 100. Sensor device 100 comprises tissue sections orother analytes 101, brass or other conductor 102, conducting tape 103,ITO or SnO2 coated slides 104, gasket insulator 105, matrix (includingbuffers) 106, microscope objective 107, CCD camera or other detector108, and voltage generator 109.

Note that the conducting tape is shown as in a semi-transparent fashion.It does not cover the edge of the sensor plates (slides) for most of thelength of the sensor. This is the portion of the sensor that will beused for TIRF illumination, should the illuminator described later beused for visualization of the analytical results. Several differenttypes of visualization can be used, as noted in the text.

FIG. 2 shows the molecular beacon for β-actin. FIG. 2 illustrates thebase sequence for a molecular beacon that can be used to recognizeβ-actin that was purchased from IDT DNA technologies. It contains arhodamine red fluorophore at its 5′ end that is quenched by a black hole2 quencher at its 3′ end in the absence of β-actin. The beacon containsa biotin moiety attached to thymidine that was introduced duringsynthesis and that enables the beacon to be bound tightly tostreptavidin. 5′Rhodamine-red-CAC-CGC-TAG-ATG-GGC-ACA-GTG-TGG-GTG-ACG-CGG-TG-BlkHoleQ2-3′.

FIG. 3 shows the steps in the preparation of biotinylated sensorsurfaces. The Steps in the preparation of biotin albumin coated sensorsurfaces are: (1) Clean ITO slides in H₂O/H₂O₂/NH₃ (10:2:0.6) 55° C. 75minutes; (2) Bake slides in vacuum oven 165° C. 150 minutes; (3) Coolwith dry nitrogen and coat with SigmaCote; (4) Coat slide with 0.05%bovine serum albumin-biotin (BSA-B) overnight; (5) Wash in phosphatebuffered saline (PBS) thoroughly; (6) Coat BSA-B treated slide withstreptavidin 0.1 mg/ml 60 minutes; (7) Wash in PBS thoroughly; (8) Coatstreptavidin treated slide with molecular beacon (0.1 nMole/ml) 60 min;and (9) Wash thoroughly.

FIGS. 4A-4B illustrate the polarization routines. FIG. 4A showsnegatively charged oligonucleotides migrating towards the positivelycharged sensor surface. The routine is suited for a sensor in whichmolecular beacons are coated to the sensor surface throughout theanalysis as in Example 1. Many other modifications of this will workalso. Much higher frequencies would normally be employed (i.e., 200,000Hz). Changes in the frequency, amplitude, and waveform alter theconcentration of the oligonucleotide in the vicinity of the sensorsurface and can facilitate or hamper hybridization. Use of voltagepatterns such as those illustrated here can be used to alter thehybridization as a function of charge and frequency. This can acceleratebinding of the analyte to the sensor surface, increase the specificityof binding interactions, and reduce the non-specific binding. FIG. 4Bshows the use of a wave form to prevent premature separation of theanalyte and the detection reagent (i.e., fluorescent PNA designed tocontain a single positive charge). This is a routine suited for a sensorin which molecular beacons are not to the sensor surface and are freeduring analysis as in Example 2. Many other modifications of this willwork also. Note the frequency shown is diagrammatic only. Much higherfrequencies would normally be employed (i.e., 200,000 Hz). This stepinvolves substantial oscillations during the binding phase followed by achange to a constant voltage to drive the complex to the anode.

FIGS. 5A-5B illustrate the principle of sensor operation in Example 2.In this mode of operation the fluorescent detection molecule is usuallyuncharged or contains a small charge that is opposite that of theanalyte. FIG. 5A shows formation of the complex. The complex has thecharge found on the analyte. Following complex formation, thefluorescent molecule is carried to one electrode, away from thenon-bound fluorophore. FIG. 5B shows that during the separation phase,the fluorescent complex migrates to the anode where it would be observedand the fluorescent unbound PNA migrates to the cathode. Its presence atthe cathode would make it invisible to an observer viewing the anodewith TIRFM.

FIGS. 6A-6B illustrate TIRF illuminator for multiple objectives. FIG. 6Ashows a side view with the position of the light source and objective.Illuminator 600 comprises a laser source 601, a lens 602, a cube 603, aprism 604, a focal point located at the junction of the prism andcoverslip 605, the surface of the sensor illuminated 606, the tissuesample 607, the surface of the sensor not illuminated 608, the holder609, and the objective 610.

The surface area illuminated on the sensor would depend on the curvatureof the cylindrical element and its distance from the sensor surface.Only the surface facing the sample would elicit fluorescence. A cutofffilter would need to be placed between the sensor and the detector todistinguish light of different colors—for example from different quantumnanodots.

FIG. 6B illustrates the manner in which the illuminator would be mountedon a microscope.

As shown, the illuminator would be held adjacent to the sensor surfacesuch that both would move side to side as a unit. The sensor could bemoved forward and backward relative to the illuminator. This wouldpermit different “slices” of the sensor to be observed.

FIGS. 7A-7B illustrate a modification of the sensor that can be used forheating. FIG. 7A is an end view of the sensor and FIG. 7B is a side viewof the sensor. The components of this figure are similar to those ofFIG. 1. The major differences involve the modifications needed toprovide the mechanism for heating. These include the second ITO layer onthe sensor slides and the insulation needed to keep the voltage that isadded to this layer from interfering with that that controls theoperation of the sensor as a measurement device.

FIG. 7A shows an end view of the sensor device 700. Sensor device 700comprises tissue sections or other analytes 701, brass or otherconductor (inner coating) 702, conducting tape 703, ITO or SnO2 coatedslides 704, gasket insulator 705, matrix (including buffers) 706,microscope objective 707, CCD camera or other detector 708, voltagegenerator 709, brass or other conductor (outer coating) 710, andinsulating tape 711.

FIG. 7B shows an end view of the sensor device 700. Sensor device 700comprises tissue sections or other analytes 701, brass or otherconductor (inner coating) 702, conducting tape 703, ITO or SnO2 coatedslides 704, gasket insulator 705, matrix (including buffers) 706,microscope objective 707, CCD camera or other detector 708, voltagegenerator 709, brass or other conductor (outer coating) 710, andinsulating tape 711.

FIG. 8 illustrates a microtiter well plate design. Microtiter well plate800 contains a top with pins 801 in electrical contact glued to a bottom802 to form wells.

Microtiter well plates that contain conducting surfaces can beconstructed in a variety of methods. The only requirement is that twoelectrically conducting surfaces be able to contact fluids within thewell. One method of constructing a plate in which all the wells will beat the same potential is shown in this FIG. 8. A plate that is coatedwith ITO or other conducting material is used as the base of themicrotiter well plate. A molded plastic adapter that forms theindividual wells is glued to the metal surface of the plate. The lowerpart of the top of the plate is made to contain pins that are fabricatedfrom plastic or other convenient material and these are coated with ITOor other metal by a sputtering process. Closure of the plate brings themetal coated pins in contact with fluids in the plate, which are incontact with the metal coated surface on the bottom of the plate.Electrodes are glued to the top and bottom coating and used to create anelectrical potential in the well.

In this arrangement, each well will be at the same electrical potential.An alternate mode of constructing the plate top can be used to createplates in which the electric potential in each well can be controlledseparately. One way of doing this is to use a top that lacks aconductive layer. A separate wire is inserted through the top into eachwell. When the microtiter plate is closed, the wire will make electricalcontact with the wells.

It should also be noted that it is not necessary for the top of themicrotiter plate to contain electrodes. To prepare a device that can beused in an open format, an electrically conducting surface is sputteredon the molded plastic layer that is used to form the walls of the wellsto completely coat its inside and outside surfaces. An insulating layeris then coated on the bottom of this molded piece before it is glued tothe metal-coated bottom.

FIG. 9A illustrates the overall design of the polymer-based device,which is shown in an expanded schematic form. The following componentsare present and identified by number. Other variations of this designare possible, however, and these are indicated by the word “optional”associated with the component. The presence of these components canfacilitate the analysis but are not absolutely required for analysis.Items 1, 2, 3, and 4 can be combined into a single device termed theanode assembly. Items 6, 7, 8, and 9 can be combined into a singledevice termed the cathode assembly. These items can be in contact withone another or separated by a fluid during operation of the device. Notethat the stippling used to mark each portion of the sensor is notintended to imply that the compositions of these portions of the sensorneed to be identical. Note also that the thickness of each layer candiffer and that it is not necessary to make them of equal thickness. Infact, it is often beneficial to make them of different thickness.

1. Electrode and electrode holder

2. Spacer to separate electrode and holder from optical surface(optional depending on the design of the electrode holder). This can bemade of a hydrogel, sintered polypropylene, or other porous substances.

2a. Semi-permeable membrane to trap analytes

3. Polymer or other material used for optical analysis

4. Polymer or other material to used as a spacer and to facilitatemixing—separates optical analysis surface from sample.

5. Sample

6. Polymer or other material used as a spacer (optional, permitsadditional analyses)

7. Polymer or other material used as a spacer (optional, permitsadditional analyses)

8. Spacer to separate electrode and holder from optical surface(optional depending on the design of the electrode holder). This can bemade of a hydrogel, sintered polypropylene, or other porous substances.

9. Electrode and holder.

FIG. 9B illustrates the device as it is being assembled. The tissuesection (5) is placed on either the lower or upper assembly, which arecomposed of components 1, 2, 3, and 4 and of components 6, 7, 8, and 9,respectively. It is usually most convenient to place it on the anodeassembly as shown here, but it does not matter which is used first. Thenthe other assembly is added to complete the device, which has all 9components as shown. Note that the components are identified in FIG. 9A.

FIG. 9C illustrates the device as it is being used duringelectrophoresis. A convenient means of doing the electrophoresis is totake the assembled components shown in FIG. 9B and slide them into a boxthat contains the connections that enable a voltage to be placed on theelectrodes (items 1 and 9 in diagrams 9A and 9B). This holds the sensordevice together and can be up-ended. This keeps any bubbles that ariseduring electrolysis of water from interfering with analysis. The boxcontains electrodes that make electrical contacts with those on theouter edges of the sensor. As noted later, it is also possible toeliminate the electrodes on the sensor or the box, but not both. Note,the components can be identified by reference to FIG. 9A. Note also, theelectrophoresis chamber is made from Plexiglas or similar plastic andcontains two vertical triangular pieces of plastic along the edgesdenoted “Anode” and “Cathode” that prevent the sensor sandwich (i.e.,the stack at the left) from being inserted into it in an incorrectorientation or if it has been assembled incorrectly from two cathodeassemblies or two anode assemblies.

FIG. 9D illustrates the construction of the anode (component #1 pluscomponent #2) and cathode (component #8 plus component #9). Both theanode and the cathode can be constructed in the same fashion but eachhas a different corner cutout as shown in the panels at the left, whichprevents them from being inserted into the electrophoresis box (FIG. 9C)in an improper orientation. The sole function of these components is todeliver a voltage across the device in a way that does not disrupt thefunctions of the gels. The solid gray rectangles indicate the metalelectrodes and the crosshatched areas indicate the plastic holder.Together, these correspond to components #1 and #9 in FIG. 9A. Theplastic holder is made from a square rod that is cutout to accommodatethe metal electrode and the sintered polyethylene frit that is stippledin these diagrams and corresponds to components #2 and #8 in FIG. 9A.The left panel illustrates cross sections of the device through theposition noted on the figure as they are modified for the anode (lowerdiagram) and cathode (upper diagram). The second, third, and fourthpanels illustrate longitudinal views from the side, front, and back.(The front is the surface that is in contact with the polymer or adialysis membrane.) Note that the device is filled with fluid before thefrit is glued to the top. This creates an air space at the top of thedevice that permits gasses to be vented caused by electrolysis duringelectrophoresis. Note that the cap is surrounded by a heat shrinkplastic coating (indicated by the black square dots) that is removedduring use of the device. This prevents loss of fluid from the deviceduring storage. Passage of fluid through the other frit is blocked bythe presence of components (i.e., #2 a and #3 or #7, FIG. 9A), whichcontact it. As a result, there is no need for the technician or otheroperator to add fluid to the device during its use. Small amounts ofdetergents can be used to facilitate wetting of the frit although thisis usually not needed. Devices can also be constructed in which thefluid is added by the operator at the time of use. Note that in thiscase, it is not necessary that the anode or cathode components #1 and #9contain the electrode (solid gray rectangles). The anode and cathodecomponents can be located in the electrophoresis box to which theoperator would add the buffer fluid. In this case, it would also not beessential to add the frit or the temporary seal to the top of the deviceas shown in the longitudinal views in this figure.

FIG. 9E illustrates the construction of the anode and cathodeassemblies. The polymeric gels that are to be included into the assemblyare prepared separately and cut into the size that will be used in thedevice. This size should be at least equal to or larger than the tissuesections or other materials to be analyzed. Indeed, it is usuallypreferable to make these 25% larger than the expected tissue sections tofacilitate placing the sections on the device during operation. Since itis possible to build the device so that multiple sections can beobserved at the same time, the size of the gel pieces to be used willdepend on the number of sections that are to be placed on the device andsubjected to electrophoresis at the same time. The final assembly stepis to hermetically seal the device in a watertight bag along with a fewdrops of water to compensate for any evaporation. A small piece of moistpaper towel can also be used for this purpose.

FIG. 9F illustrates the mounting of the “exposed” sensor sandwich on thecamera. The anode and the sintered polyethylene component are removed.This is easily done by placing a small spatula between the corners ofcomponent #2 and the dialysis tubing or component #3 and twisting todislodge the anode. Care should be taken not to dislodge the dialysistubing or component #3. The remainder of the sandwich is placed on afiber optic window or a fiber optic taper that is coupled to the chip ofa sensitive CCD camera (11). The sample will be detected by totalinternal reflection fluorescence (TIRFM) using an illumination systembased on a laser or other illumination device that illuminates component#3. Although not depicted, the illumination is designed such that theentire area of the face of this component is illuminated. This willenable the CCD camera to record an image of the entire section at onetime. The resolution of the camera will depend on the number of pixelson its chip, the size of each pixel, and the sizes of the fibers thatare used to make the fiber optic window or taper. A resolution ofbetween 20-50 μm is sufficient for the analysis since this will enablethe determination of the RNA to an area of 2-3 cells. This informationis transferred to a computer for data processing. The cathode can alsobe removed if desired, but this is not essential until the tissue sliceis to be examined by regular microscopy. This will often depend on whatis seen from the fluorescent image.

FIG. 10A illustrates the migration of PNA labeled with a fluorophore(PNA*) when it is free and bound to RNA in the sensor apparatus. Notethat the complex will not pass through the dialysis membrane (component#2 a) due to the limitation of the pore size. The pore size of component#3 can also be kept small such that the RNA/PNA* complex will notpenetrate through it in which case the dialysis membrane component(i.e., #2 a) is not essential and would not be used. It is critical thatthe uncomplexed PNA* not enter the compartment created by component #3,however, since this would create an unacceptably high background in thedevice. Thus, it is important to use PNA* that are positively charged inthe vicinity of component #3 so that they migrate away from thiscomponent and from its surface. Since the analysis will take advantageof the principle of total internal reflection fluorescence (TIRF), inwhich materials that are outside the standing evanescent wave that iscreated by illumination of component #3, the distance of the PNA* fromcomponent #3 need be only a few hundred nanometers.

FIG. 10B illustrates the migration of a fluorescent charged detectionagent before and after its charges have been removed by an enzyme or areaction with materials in or released from the tissue section. When theunreacted detection is positively charged and the reaction causes it tobecome negatively charged by removing its positively charged residuessuch as lysine or arginine amino acids, this would change the charge onthe detection agent and cause it to migrate towards component #3 asshown. Furthermore, the fluorescent detection agent could be designedsuch that removal of the charged portion exposes a binding site thatwill interact with a site or sites coupled to component #3. Thus, it canbe seen that a change in the charge of a detection agent or theformation of a complex that has a different charge from the uncomplexeddetection agent can be used for the detection of analytes, includingthose that are spatially organized such as would occur in tissuesections.

FIG. 11 illustrates design considerations for component #3. Thecross-linking of the hydrogel in component #3 should depend on theanalysis. In the case of analytes such as RNA, it is often useful toemploy a high cross-linking, which will keep the refractive index highand cause the hydrogel to behave as a semi-permeable barrier to RNA,keeping it on the surface that faces component #4. Also, in the case ofnegatively charged analytes such as RNA that are to be detected withpositively charged reagents such as PNA that contain positively chargedresidues, the surface of component #3 can be cross-linked with apositively charged material that will repel the detection reagent unlessit is bound to the negatively charged RNA. This will also be facilitatedby using a buffer that has a pH that is lower than that of the pI of thePNA. The surface of component #3 that faces component #2 should behydrophilic to make it attract an aqueous layer that can be used toseparate it from the fiber optic.

FIG. 12A illustrates the arrangement of the system used to illuminatecomponent #3 (or component #7, when used). Component #3 is placed on topof the optical fiber such that a thin water or buffer layer separatesthe two. This is needed to cause total internal reflection of theillumination beam (heavy black arrow). Fluorescence (thin downwardpointing arrow) passes through the buffer layer and through a filter (ifpresent) that is designed to block scattered illumination. Thisillumination should be minimal when the interfaces of components #3 and#4 and the water and component #3 are smooth and clean. Use of a cutofffilter can reduce scattered light but will make it more difficult tomeasure the emission at more than one excitation wavelength unless afilter wheel assembly is employed. A useful way to increase the signalto noise ratio is to illuminate the sample with polarized light and toblock the transmission of light having this polarization with a filterat the location shown. Note that the laser beam should be compressed inthe vertical direction and expanded in the horizontal direction toenable illumination of the entire surface of component #3. This willpermit an image of the analyte in the entire tissue section depicted ascomponent #5 is to be determined at one time. Note that the size of thesensor as reflected in component #3 should be slightly smaller than thesize of the image that is taken from the fiber optic. This is to permita low-resolution image to be taken of the outline of component #3. Thiscan be used to align the fluorescence image with that of the tissuesection when the sensor sandwich is transferred to a standard invertedmicroscope for observation through an objective.

FIG. 12B illustrates the illumination used to distinguish colors. Thefilled triangles indicate the relative wavelength used for illuminationwith the right most position indicating longer wavelengths and the leftmost position indicating shorter wavelengths. Component #3 is firstilluminated with the longest wavelength and the fluorescence measured.It is then illuminated sequentially with increasingly short wavelengthsas represented by the panels going from the top of the figure to thebottom of the figure. The fluorescence excitation spectrum isrepresented by the dotted black lines in each panel. The fluorescenceemission spectrum is represented by the dashed gray lines in each panel.The fluorescence that is measured is represented by the solid blacklines. As is represented schematically here, the increase in totalfluorescence represented by the black lines at increasingly shorterwavelengths can be resolved mathematically by “subtracting” thefluorescence from each of the subsequent lines. This is done via amatrix algebra approach in which the fluorescence excitation andemission standards is known at each wavelength employed.

FIG. 12C illustrates a preferred type of filter that can be used in thedevice to permit distinguishing colored fluorophores, if it is necessaryto reduce the amount of scattered light. This type of filter is known asa multi-band pass filter because it has the ability to block wavelengthsof several laser lines such as those indicated by the broken lines underthe letters B, G, and R. As a result scattered light that is used toexcite the sample by total internal reflection will be prevented fromreaching the fiber optic window or fiber optic taper and will notinterfere with analysis. In the diagram below, the B, G, and R refer tothe maximum emission of blue, green, and red lasers respectively. Sincethis is an emission filter, it would also reduce the amount offluorescent signal but it would increase the signal to noise ratio byreducing the amount of scattered light even further. A second type offilter that could be used blocks polarized light. Since light emitted byfluorophores that are illuminated by evanescent light will not have thesame polarity as the light used to illuminate component #3, the lightthey emit will not be blocked by a filter that is designed to blockpolarized light that is used for illumination. Therefore, thepolarization filters will block the light scattering much moreeffectively than they will block the fluorescent signal. This will raisethe signal to noise ration. Finally, a third means of distinguishingcolor in this device is to employ fluorophores that are photobleached atdifferent rates. By monitoring the change in signal as a function oftime, it is possible to distinguish each of the fluorophores. This alsopermits use of fluorophores that have nearly identical emission spectra.Thus, fluorescence from a fluorophore that is readily photobleached willdecay much more rapidly than that from a fluorophore that is morestable. When this type of analysis is employed, it is desirable to uselabel the more abundant analytes with the fluorophore that is the leaststable.

FIG. 12D illustrates a preferred mode for illuminating the sample.Illumination of the sample can be accomplished using a fiber opticbundle that is divided into fibers that are held in a linear array nextto component #3 as shown here looking down at component #3. The diagramalso shows that more than one fiber bundle can be used if desired. Thiscan be connected to the same laser(s) indicated on the figure, or it canbe connected to different lasers. Note that the number of fibers shownon this diagram is for illustration purposes only. There can be feweror, more likely, many more fibers. The diameter of the fibers (core pluscladding) should be less than the thickness of component #3. Thenumerical aperture of the fibers should be chosen to be smaller thanthat which violates the principle of total internal reflection. Thiswill depend on the refractive index of component #3 and the refractiveindices of the materials above and below component #3 that contact it.This angle can be calculated from the Snell equation.

One aspect of the invention provides hydrogels similar to those used tomake contact lenses that can be used in a sensor because the hydrogelsare suitable for electrophoresis and optical refraction and capture ofreagents. The other aspect of the invention is the sensor itself andwill depend on how the sensor is used. The sensor is designed to be userfriendly in that the user does not need to add any fluids. For thisreason, the electrodes need to be built into the sensor. In other uses,the user can add the fluids. In this case the electrodes do not need tobe built into the sensor per se, but can be built into theelectrophoresis box. FIG. 9 shows them simply to make electrical contactwith the electrodes in the sensor device. If one were to add fluid tothe box, then the electrodes would not need to be in the sensor. Anotheraspect of the invention is that the charge of the material doing theanalysis is altered during analysis. This change in charge occurredbecause the detection agent became bound to the analyte (i.e., the PNAare designed to be positively charged and the complex with RNA will benegatively charged). It is also possible for the detection agent to bemodified by the analyte and to have its charge changed. Thus, an enzymethat cuts off a positively charged portion of the analyte can alter itscharge. This will cause it to migrate towards the anode if this resultsin a change from positive to negative. This can also be used to create anew binding surface on the analyte as well.

The word “bound” reflects the idea of “change” as well as “binding.”Interaction of the detection reagent with the analyte leads to a changein the direction of its migration in an electric field. Electrodes donot need to be attached to the sensor per se unless the device is to beconstructed such that the user does not need to add fluid. A spacerwould still be required to keep the component #3 from touching theelectrode to permit bubbles to escape the device. The device as shown isuseful for analyses that are located at different spatial positions inan analyte such as a tissue section.

REFERENCES

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Throughout this application, various publications have been referenced.The disclosures in these publications are incorporated herein byreference in order to more fully describe the state of the art.

While the invention has been particularly described in terms of specificembodiments, those skilled in the art will understand in view of thepresent disclosure that numerous variations and modifications upon theinvention are now enabled, which variations and modifications are not tobe regarded as a departure from the spirit and scope of the invention.Accordingly, the invention is to be broadly construed and limited onlyby the scope and spirit of the following claims.

1-14. (canceled)
 15. A method for detecting an ionic analyte in a samplein which an analyte is bound to a detection reagent to form a boundcomplex, comprising the steps of: (A) providing a sensor devicecomprising: (a) a sample (5) comprising an ionic analyte and a detectionreagent in a conductive fluid, wherein the detection reagent has a netcharge different from the analyte; (b) a first permeable polymerichydrogel plate (3) and a first spacer plate (8), which plates provide acompartment for the sample; (c) an anode (1) juxtaposed to the outsideof the first hydrogel plate and not in contact with the sample; (d) acathode (9) juxtaposed to the outside of the first spacer plate and notin contact with the sample; (e) a voltage generator (10) to apply anelectric potential to the anode and cathode; and (f) a detector (11);and (B) adding the ionic analyte and detection reagent in the conductivefluid to the compartment; (C) applying an electrical potential via thevoltage generator; and (D) detecting via the detector the bound complexformed from the analyte because the bound complex has a charge thatcauses it to migrate in a direction opposite from that of the unboundanalyte when the electric potential is applied.
 16. The method accordingto claim 15, wherein the ionic analyte is a gene product in a cell ortissue section sample.
 17. The method according to claim 16, wherein thegene product is a nucleic acid or protein.
 18. The method according toclaim 15, wherein the detection reagent is selected from the groupconsisting of uncharged peptide nucleic acids, negatively chargedpeptide nucleic acids, positively charged peptide nucleic acids, peptidenucleic acids labeled with a fluorophore, and peptide nucleic acids inwhich a phosphate group has been replaced by a sulfate group or acarbonate group.
 19. The method according to claim 18, wherein thepeptide nucleic acid has a hairpin conformation.
 20. The methodaccording to claim 15, wherein the first permeable polymeric hydrogelplate comprises a hydroxyethylmethacrylate or ahydroxyethylmethacrylate-methacrylic acid.
 21. The method according toclaim 15, further comprising a second permeable polymeric hydrogel plate(7) juxtaposed between the first spacer plate and the sample.
 22. Themethod according to claim 21, wherein the second permeable polymerichydrogel plate comprises a hydroxyethylmethacrylate or ahydroxyethylmethacrylate-methacrylic acid.
 23. The method according toclaim 15, further comprising a second spacer plate (2) juxtaposedbetween the first permeable polymeric hydrogel plate and the anode. 24.The method according to claim 23, further comprising a semipermeablemembrane (2 a) juxtaposed between the second spacer plate and the firstpermeable polymeric hydrogel plate.
 25. The method according to claim21, further comprising a first (4) polymeric plate juxtaposed betweenthe first permeable polymeric hydrogel plate and the sample and a second(6) polymeric plate juxtaposed between the second permeable polymerichydrogel plate and the sample, wherein the first and second polymericplates have a lower refractive index than that of the first and secondpermeable polymeric hydrogel plates, respectively.
 26. The methodaccording to claim 25, wherein the first and second polymeric platescomprise a polyacrylamide, an agarose gel, a hydroxyethylmethacrylate,or a hydroxyethylmethacrylate-methacrylic acid.
 27. The method accordingto claim 15, wherein the detector is a fluorescence, luminescence,colorimetry, or total internal reflection illumination detector.
 28. Themethod according to claim 15, wherein the detector detects by phasecontrast microscopy, bright field microscopy, darkfield microscopy,differential interference contrast microscopy, confocal microscopy, orepifluorescence microscopy.
 29. The method according to claim 15,wherein the electrical potential is applied perpendicular to the platesand is constant or varied such that the overall effect is to have eachplate have a net charge, such that charged analytes in the sample willmigrate to one plate.
 30. The method according to claim 15, wherein theelectrical potential is applied perpendicular to the plate ands isalternated such that there is no net charge on either plate, such thatcharged analytes will oscillate back and forth in the central space awayfrom either plate where they interact with the detection reagent. 31.The method according to claim 15, wherein the detection reagent is amolecular beacon.
 32. The method according to claim 15, wherein a secondmolecular beacon is employed as an internal control.
 33. The methodaccording to claim 32, wherein a first molecular beacon is employed todetect a control gene product and a second molecular beacon is employedto detect a gene product of experimental or diagnostic interest, whereinthe first and second molecular beacons are each labeled with a differentfluorophore that emits at a different wavelength so that the first andsecond molecular beacons can be simultaneously analyzed.